Methods for detecting intermolecular interactions in vivo and in vitro

ABSTRACT

Methods for assessing intermolecular interactions in vivo and in vitro are provided. Methods are provided for detecting protein-DNA interactions in vivo, in which a cell having a chimeric guide endonuclease molecule and a target nucleic acid is provided, and cleavage of the target nucleic acid by the chimeric guide endonuclease molecule is monitored. Cleavage by the chimeric guide molecule corresponds to binding of the guide domain to the target nucleic acid, or to a protein associated with the nucleic acid. The methods of the invention are adapted to cleavage of target nucleic acids, amplification of target nucleic acids, detection of target nucleic acids, screening of genomic target nucleic acid sequences for guide binding domains, and screening for modulators of chimeric guide binding domain activity. Also provided are methods for detecting interactions between other molecules, including hormones and receptors, enzymes and substrates, and the like.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. patent application Ser.No. 08/826,622, filed Apr. 3, 1997, which was converted to provisionalapplication Ser. No. ______ by way of petition filed on Nov. 19, 1997.This application is also related to co-filed U.S. patent application byJay Chung entitled “Chimeric Endonucleases for Detecting Protein-nucleicAcid Interaction In Vivo and In Vitro,” filed Apr. 3, 1997, Ser. No.08/825,664, which was converted to provisional application Ser No.______by way of petition filed on Nov. 19, 1997, and to co-filed patentapplication Ser. No. ______ by Jay Chung entitled “ChimericEndonucleases For Detecting Intermolecular Interactions In Vivo And InVitro”, filed on Apr. 2, 1998 as Attorney Docket No. 15280-31820US.These applications are incorporated by reference in their entireties forall purposes.

FIELD OF THE INVENTION

[0002] This invention pertains to the field of detecting intermolecularinteractions in vivo and in vitro using chimeric endonucleases thatinclude a nuclease cleavage domain that is linked to a moiety that iscapable of directly or indirectly binding to a molecule of interest. Theprovided chimeric endonucleases are useful for determining the locationat which a protein becomes associated with a nucleic acid or othermolecule.

BACKGROUND OF THE INVENTION

[0003] Molecular interactions form the basis of many biological andchemical events. These include enzymatic reactions, hormone-ligandinteractions, drug or toxin-protein interaction and otherprotein-protein, protein-nucleic acid, and nucleic acid-nucleic acidinteractions. Therefore, in order to understand these biological andchemical events, the ability to detect contact or close proximitybetween two known molecules would be of great value.

[0004] The techniques to visualize a protein complex formed on aparticular nucleic acids sequence, such as the electrophoretic mobilityshift assay (EMSA) and footprinting assays, in vitro and in vivo, havebeen crucial to understanding how transcription occurs. Presenttechniques, however, have serious limitations. In a living cell,transcription happens in a far more complex environment than can beduplicated in vitro and as a result, in vitro techniques will not alwaysdepict accurately the situation in vivo. For example, in vitrotechniques have not been useful in studying long range interactions (>1kb) such as that of the β-globin LCR which play an important role intranscription in a living cell. In vivo footprinting, on the other hand,reflects protein-DNA interactions in a living cell, but the identity ofthe complex creating the footprint is not known.

[0005] Hybrid affinity cleaving proteins composed of a DNA bindingdomain of a protein and a Fok 1 endonuclease have been used in vitro forcleavage of nucleic acids in a variation of in vitro footprintingmethods. See, Chandrasegaran, U.S. Pat. Nos. 5,487,994 and 5,436,150;Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160 and Kim etal. Proc. Natl. Acad. Sci. USA (1994) 91: 883-887. However, here again,it has typically been assumed that the DNA binding domain is sufficientto confer sequence specific binding, and therefore only the DNA bindingdomain of the transcription factor has been included in the hybridprotein. In vitro, this assumption may be correct, since there are noother proteins present when the hybrid protein binds to the DNA.However, this assumption may not apply in vivo, and may not provide arealistic assessment of the nature of an in vivo DNA-protein complex.Furthermore, these methods do not provide any way of assessing theinteraction of proteins which bind indirectly to a nucleic acid, such asproteins which form a transcription initiation complex.

[0006] Previously available methods of detecting interactions betweenmolecules in the context of cells or organisms have been hampered by alack of resolution and insufficient sensitivity. With the current stateof the art technique, which combines confocal microscopy andimmunofluorescence, two molecular species can be visualizedsimultaneously. If the two molecular species interact, their fluorescentsignals should colocalize. However, because the limit of the resolutionfor light microscopes is in the order of microns, the two molecularspecies could be quite far apart in molecular terms and still appear tocolocalize. In addition, because fluorophores are directly conjugated tothe antibodies in conventional immunofluorescence, there is no way toamplify the signal in a manner analogous to the amplification thatoccurs when horse radish peroxidase or alkaline phosphatase conjugatedantibodies are used. As a result, the sensitivity of the conventionalimmunofluorescence is limited.

[0007] New techniques which provide ways of assessing in vivo nucleicacid-protein, protein-protein, and other intermolecular interactionswould be desirable, because they would provide new tools for studyinggene regulation, enzymatic reactions, hormone-receptor interactions, andother cellular processes in vivo. In addition to the value that such newmethods would bring to basic research, the ability to assess accuratelyin vivo nucleic acid-protein interactions could provide tools forisolating genes, identifying transcription factors, identifyingregulatory regions, identifying transcription factor modulating agentsand the like. The present invention provides, inter alia, new methodsfor assessing in vivo DNA-protein interactions, in vivo RNA-proteininteractions, indirect protein-nucleic acid interactions and the like,thereby providing new tools for studying gene regulation, isolatinggenes, identifying transcription factors, identifying transcriptionfactor modulating agents, identifying regulatory regions and many otherfeatures which will become apparent upon further reading. Also providedare new methods for visualizing other types of intermolecularinteractions with high resolution and sensitivity.

SUMMARY OF THE INVENTION

[0008] The present invention provides methods which provide in vivoprocedures for assessing protein-nucleic acid interactions. Thesemethods represent a novel technology referred to generally as ProteinPosition Identification with Nuclease Tail or “PINPOINT” methods. In themethods, a fusion partner which binds directly or indirectly to a targetnucleic acid is fused, typically via a linker, to an endonuclease domainwhich cleaves the target nucleic acid. By monitoring the in vivocleavage of the target nucleic acid by the endonuclease domain, it ispossible to quantitatively and qualitatively monitor interactionsbetween proteins and nucleic acids, between protein complexes andnucleic acids, and between individual members of a protein complex and anucleic acid. These methods are adapted to basic research, drugscreening methods, methods of finding targets for transcription factorssuch as oncogenes, methods of finding transcription factors for targetsequences, and the like.

[0009] The present invention further provides methods of screening testnucleic acids for in vivo binding sites which are cleaved by a chimericguide endonuclease fision molecule. Typically, a cell comprising achimeric nucleic acid encoding the chimeric molecule and a test nucleicacid is provided, the chimeric nucleic acid is expressed in the cell,thereby producing a chimeric guide protein in the cell and, the cell isincubated under conditions in which the guide protein is active.

[0010] In one assay of the invention, the test nucleic acid includes apromoter sequence operably linked to a reporter gene. Detection of thepresence or absence of reporter gene expression is an indicator forwhether the test nucleic acid comprises an in vivo binding site for thechimeric guide molecule. In one class of embodiments, the cell isprovided by co-transducing the cell with a plasmid encoding the targetnucleic acid and a plasmid encoding a chimeric guide protein.Optionally, one or more additional plasmids comprising one or moreadditional test nucleic acids are also transduced into the cell, and theeffect of the chimeric guide molecule is assessed simultaneously on morethan one test nucleic acid.

[0011] Parallel screening formats are provided, in which a second cellcomprising a second chimeric nucleic acid encoding a second chimericguide molecule and a second test nucleic acid is provided. The secondchimeric nucleic acid is expressed and the effect of the chimericnucleic acid on the second test nucleic acid is monitored.

[0012] The invention provides methods of detecting and assessing anucleic acid binding molecule modulating agent. In the methods, a cellcomprising a test nucleic acid binding site (e.g., a promoter sequencewhich is bound by a transcription factor) and a chimericguide-endonuclease molecule is provided. The cell is contacted with thepotential modulating agent, and the rate of cleavage of the test nucleicacid binding site by the chimeric molecule in the presence of the agentin measured.

[0013] In one class of embodiments, the invention provides methods ofcleaving target nucleic acids in vitro or in vivo. In the methods, atarget nucleic acid is contacted by a guide-micrococcal endonucleasefusion molecule in the presence of calcium. The guide-micrococcalendonuclease fusion then cleaves the target nucleic acid. In oneembodiment, the cleavage is performed in situ, e.g., in a tissue or cellsample on a solid substrate such as a microscope slide.

[0014] The present invention also provides methods for assessingintermolecular interactions both in vivo and in vitro. These methodsrepresent a novel technology referred to generally as “FLASHPOINT”methods. In these methods, which are useful for detecting whether afirst molecule is in close proximity to a second molecule, a molecularbeacon is attached to the first molecule. The molecular beacon, whichcan be attached directly or indirectly to the first molecule, istypically an oligonucleotide to which is attached a fluorophore and aquencher. A chimeric endonuclease is attached to the second molecule,either directly or indirectly. To determine whether the first moleculeis in close proximity to the second molecule, one detects whetherfluorescence is emitted by the fluorophore. Fluorescence emission isindicative of cleavage of the oligonucleotide by the endonucleasemoiety, thereby causing separation of the fluorophore and the quencher.In a preferred embodiment, the endonuclease moiety is inducible (e.g.,calcium inducible).

[0015] In another embodiment, the invention provides methods ofobtaining increased sensitivity in assays such as immunoassays. Themethods involve detecting a target molecule by contacting the targetmolecule with a chimeric endonuclease. The target molecule and thechimeric endonuclease are placed under conditions conducive to formationof an association between the target molecule and the chimericendonuclease. The chimeric endonuclease is then contacted with amolecular beacon that is composed of an oligonucleotide to which isattached a fluorophore and a quencher. The presence or absence of afluorescent signal is then detected. A signal, if present, results fromcleavage of the oligonucleotide by the endonuclease, which causesseparation of the quencher from the fluorophore.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a schematic drawing of a method of detecting a plasmidtarget nucleic acid in vivo.

[0017]FIG. 2 is a schematic drawing of a method of detecting a genomictarget nucleic acid in vivo.

[0018]FIG. 3 is a schematic drawing of a method of detecting an unknowngenomic target nucleic acid in vivo.

[0019]FIG. 4 is a schematic drawing of a method of making a directory ofaddresses of unknown genomic target nucleic acids in vivo.

[0020]FIG. 5 is a schematic drawing of a method of detecting an RNAtarget nucleic acid in vivo.

[0021]FIG. 6 is a schematic drawing of a method for detecting targetnucleic acids using a chimeric protein comprising an immunoglobulin.

[0022]FIG. 7 is a schematic diagram of the FLASHPOINT strategy. Thestructure of molecular beacon is shown on the left. In the loop portion,the molecular beacon contains an amino-modified (NH₂) thymidine (T) andA-T rich nucleotides (thick line). A reporter fluorophore (filledcircle) that is excited by UV light and a quencher (gray circle) arepresent at the 5′ and 3′ ends of the oligonucleotide, respectively. Thenuclease cleaves preferentially in the A-T rich region resulting in theremoval of the quencher and emission of fluorescence by the reporterfluorophore.

[0023]FIG. 8 shows one application of FLASHPOINT to detect proximity ofmolecules A and B. The position of molecule A is marked by the antibodyagainst molecule A which is crosslinked to the nuclease tail throughSMCC (IMMUNOPOINTER), and the position of molecule B is marked by anantibody against molecule B that is crosslinked to the molecular beaconthrough BS3. If molecules A and B are separated by less than 10 nm, themolecular beacon is cleaved by the nuclease and the reporter fluorophoreemits fluorescence (below).

[0024]FIG. 9 shows the fluorescence amplification method provided by theinvention. The IMMUNOPOINTER against molecule A cleaves free molecularbeacon (substrate) resulting in the accumulation of fluorescenceemitting reporter.

DEFINITIONS

[0025] A “chimeric guide endonuclease fusion molecule” is a moleculewith an endonuclease activity domain fused to a guide domain whichrecognizes either a site on a target nucleic acid, or a site on amolecule such as a protein which binds (directly or indirectly) to atarget nucleic acid. The guide and endonuclease domains are optionallyseparated by a linker region. Guide domains are often polypeptides, butare optionally other molecules which bind directly or indirectly to atarget nucleic acid. For example, the guide domain is optionally anucleic acid which hybridizes to the target nucleic acid. Typical guidedomains are derived, inter alia from DNA binding proteins, RNA bindingproteins, proteins which bind to DNA binding proteins, proteins whichbind to RNA binding proteins, antibody proteins which binds to DNAbinding proteins, antibody proteins which bind to antibody proteinswhich bind to a nucleic acid binding proteins, and the like. Anendonuclease domain is typically a polypeptide sequence withendonuclease activity, although other molecules with endonucleaseactivity (such as RNA ribozymes) can also be used. Preferredendonuclease domains are inducible, rather than constitutive. Examplesinclude micrococcal nuclease and micrococcal endonuclease mutants whichhave lowered constitutive activity relative to a wild-type micrococcalendonuclease enzyme. In some embodiments, endonuclease domains arerecombinantly derived from naturally occurring proteins which haveendonuclease activity and are recombinantly fused to the guide domain bymaking a nucleic acid encoding the endonuclease domain and a nucleicacid encoding the guide domain, and expressing the nucleic acid. Inother embodiments, the chimeric guide endonuclease is made by chemicallycoupling the guide domain and the endonuclease domain. A chimeric guideendonuclease fusion protein is also referred to as a “pointer,” for itsability to create a cleavage site or “point” in the target nucleic acid.

[0026] The term “nucleic acid” refers to a deoxyribonucleotide (DNA) orribonucleotide (RNA) polymer in either single- or double-stranded form,and unless otherwise limited, encompasses known analogues of naturalnucleotides that hybridize to nucleic acids in a manner similar tonaturally occurring nucleotides. Unless otherwise indicated, aparticular nucleic acid sequence optionally includes the complementarysequence thereof. A “target nucleic acid” is a nucleic acid to becleaved by a chimeric guide-endonuclease fusion molecule. The cleavedtarget nucleic acid is optionally a DNA an RNA, a combination thereof,or an analogue thereof, and can be in single-stranded or double strandedform, and the cleaved nucleic acid can be modified by a polymerase, byamplification, by ligation of oligonucleotides, or the like. Essentiallyany nucleic acid can be a target nucleic acid, including chromosomalsequences, extragenomic DNA, plasmids, genomic sequences and the like.The sequence of the target nucleic acid can be known or unknown. Thecleavage site made by a chimeric guide endonuclease fusion molecule onthe target nucleic acid is referred to as a “target cleavage site” or a“point” i.e., in reference to cleavage by a “pointer.”

[0027] A cell is “transduced” with a nucleic acid when the nucleic acidis introduced into the cell. A cell is “stably transduced” with anucleic acid when the nucleic acid is stably replicated in a populationof cells.

[0028] Two nucleic acids are “ligated” together when one or morecovalent bond is formed between the nucleic acids. Ordinarily, nucleicacids are ligated enzymatically, i.e., using a ligase enzyme; however,they are optionally ligated using chemical reagents.

[0029] A nucleic acid “tag” is a short nucleic acid of known sequencewhich is ligated to one or more nucleic acids. A nucleic acid with aligated tag is a “tagged nucleic acid.”

[0030] An “internal primer” is a primer (a single-stranded nucleic acidwhich is typically between 8 and 100 nucleotides in length, usuallybetween 12 and 40 nucleotides in length and often between 17 and 30nucleotides in length) which hybridizes to a subsequence found betweenthe ends of a nucleic acid.

[0031] A “nucleic acid template” or “template” is a nucleic acid whichis copied, or, when single stranded, a nucleic acid which is used tomake a complementary nucleic acid.

[0032] A “terminal region” of a nucleic acid refers to a subsequence ofthe nucleic acid which is located adjacent to either the 5′ or 3′ end ofthe nucleic acid.

[0033] A “marker nucleic acid” is a nucleic acid encoding a selectablecomponent. A selectable component is a protein or nucleic acid whicheither increases or decreases the survival of a cell under selectedconditions, or which provides a detectable label which can be used toisolate or.identify the cell. For example, the component can encode acomponent which permits the cell to survive under controlledenvironmental conditions (e.g., an antibiotic resistance gene permits acell to replicate in the presence of the antibiotic). Alternatively, thecomponent can be a molecular tag which facilitates isolation and/oridentification of the cell (e.g., a fluorescent protein such as greenfluorescent protein (GFP) which permits separation of cells expressingthe protein using a fluorescence activated cell sorter (FACS) machine,or using HOOK selection, available from Clontech).

[0034] A nucleic acid is “amplified” when corresponding RNA or DNAnucleic acid copies of all or a portion of the nucleic acid are made.The copies of an RNA are optionally RNA or corresponding DNA, and thecopies of a DNA are optionally DNA copies or corresponding RNA copies.Copies are made using in vitro techniques such as PCR, LCR, replicasemediated replication, reverse transcription or the like, or are made bycloning the nucleic acid in a cell.

[0035] A “calcium inducible” enzyme is an enzyme which has increasedactivity in the presence of calcium. Typically, the activity increasesat least 100% in the presence of calcium, generally at least 500%,commonly 1,000% percent and often at least about 10,000%.

[0036] A “micrococcal nuclease” domain is a polypeptide derived from thecleavage domain of micrococcal (or “staphylococcal”) nuclease. Thepolypeptide will have sequence similarity to the naturally occurringnuclease enzyme, but optionally comprises deletions, insertions or othermutations which modulate the activity of the enzyme. Many suchmodifications of wild-type micrococcal nuclease are known. Aparticularly useful micrococcal nuclease domain has lower constitutiveactivity than the wild-type enzyme. Micrococcal endonuclease produces a5′ OH and a 3′ P at the site of cleavage by the enzyme. Although theenzyme typically produces single-stranded nicks in a double-strandedDNA, the enzyme also produces double stranded cuts in the DNA under somereaction conditions. Reviews of micrococcal nuclease activity includeTucker et al. (1978) Mol. Cell Biochem. 22(2-3):67-77 and Tucker et al.(1979) Mol. Cell Biochem. 23(1):3-16. Micrococcal endonuclease mutantsare described by Serpersu et al. (1987) Biochemistry 26: 1289; Serpersuet al. (1986) Biochemistry 25:68-77; Serpersu et al. (1989) Biochemistry28:1539-1548 and Serpersu et al. (1990) Biochemistry 29:8632-8642.Sequences of micrococcal nuclease are found in available databases, andin, e.g., Shortle (1983) Gene 22: 181-189.

[0037] A “class I restriction site” is a site on a nucleic acid which,when in double-stranded form, is recognized by a class I restrictionendonuclease. A “class IIS restriction site” is a site on a nucleic acidwhich, when in double-stranded form, is recognized by a class IISrestriction endonuclease. Descriptions of Class I and Class IISrestriction enzymes are found in Berger supra at chapter 11 and inSambrook and Ausubel, both infra. A Class IIS restriction enzyme cleavesa nucleic acid at a site separate from the recognition site for theenzyme. A class II recognition site is a site on a nucleic acid which,when in double-stranded form, is recognized by a class II restrictionendonuclease.

[0038] Nucleic acids are “concatemerized” when similar or identicalnucleic acids are joined, covalently, or non-covalently.

[0039] An “oligonucleotide” is a nucleic acid (DNA, RNA or analoguethereof) in single stranded or double stranded form. Typically, theoligonucleotide is less than about 100 nucleotides in length, althoughlonger nucleic acids are also considered oligonucleotides for purposesof this disclosure.

[0040] A “recombinant nucleic acid” comprises or is encoded by one ormore nucleic acids which are derived from a nucleic acid which wasartificially constructed. For example, the nucleic acid can comprise orbe encoded by a cloned nucleic acid formed by joining heterologousnucleic acids as taught, e.g., in Berger and Kimmel, Guide to MolecularCloning Techniques, Methods in Enzymology volume 152 Academic Press,Inc., San Diego, Calif. (Berger) and in Sambrook et al. (1989) MolecularCloning—A Laboratory Manual (2nd ed.) Vol. 1-3 (Sambrook).Alternatively, the nucleic acid can be synthesized chemically.

[0041] A “primer extension reaction,” is performed by hybridizing aprimer to a template nucleic acid, and covalently linking nucleotides tothe primer such that the added nucleotides are complementary to thetemplate nucleic acid. Primer extension is ordinarily performed using anenzyme such as a DNA polymerase. Using appropriate buffers, pH, saltsand nucleotide triphosphates, a template dependant polymerase such a DNApolymerase I (or the Klenow fragment thereof), taq or rTth polymerase XLincorporates a nucleotide complementary to the template strand on the 3′end of a primer which is hybridized to the template.

[0042] An “amplification primer” is a nucleic acid primer used forprimer extension in a PCR reaction.

[0043] A “region” of a nucleic acid refers to the general areasurrounding a structural feature of the nucleic acid, such as thetermini of the molecule, an incorporated residue, or a specificsubsequence.

[0044] A “restriction endonuclease cleavage site” denotes the site atwhich a known endonuclease cleaves DNA under defined environmentalconditions.

[0045] A “restriction endonuclease recognition site” denotes the DNAsite which is recognized by the endonuclease which brings about thecleavage reaction. The site is optionally from any restriction enzyme,including class I, class II, class IIS, class III, and class IV enzymes.Berger supra at chapter 11 describes several restriction enzymes. Seealso, Sambrook and Ausubel, both supra. The recognition site is distinctfrom the cleavage site for some enzymes, such as HphI and Bsg1.

[0046] An “antibody” is a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof. Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively. An exemplar immunoglobulin(antibody) structural unit comprises a tetramer. Each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). TheN-terminus of each chain defines a variable region of about 100 to 110or more amino acids primarily responsible for antigen recognition. Theterms variable light chain (VL) and variable heavy chain (VH) refer tothese light and heavy chains respectively. Antibodies exist e.g., asintact immunoglobulins or as a number of well characterized fragmentsproduced by digestion with various peptidases. Thus, for example, pepsindigests an antibody below the disulfide linkages in the hinge region toproduce F(ab)′₂, a dimer of Fab which itself is a light chain joined toV_(H)-C_(H)1 by a disulfide bond. The F(ab)′₂ may be reduced under mildconditions to break the disulfide linkage in the hinge region therebyconverting the F(ab)′₂ dimer into an Fab′ monomer. The Fab′ monomer isessentially an Fab with part of the hinge region (see, FundamentalImmunology, Third Edition, W. E. Paul, ed., Raven Press, N.Y. (1993),which is incorporated herein by reference, for a more detaileddescription of other antibody fragments). While various antibodyfragments are defined in terms of the digestion of an intact antibody,one of skill will appreciate that such Fab′ fragments may be synthesizedde novo either chemically or by utilizing recombinant DNA methodology.Thus, the term antibody, as used herein, also includes antibodyfragments either produced by the modification of whole antibodies orthose synthesized de novo using recombinant DNA methodologies.Immunoglobulins generated using recombinant expression libraries arealso antibodies for purposes of this invention. Many antibodies areavailable, and methods of making antibodies are well known. See, Paul,id. and, e.g., Coligan (1991) Current Protocols in ImmunologyWiley/Greene, NY; and Harlow and Lane (1989) Antibodies: A LaboratoryManual Cold Spring Harbor Press, NY).

[0047] A “molecular beacon” is an oligonucleotide to which is attached afluorescent moiety and a quenching moiety. When the fluorescent moietyand the quenching moiety are in close proximity, no fluorescent signalis emitted. However, when the quenching moiety and the fluorescentmoiety are no longer in close proximity, quenching does not occur and afluorescent signal is emitted. Thus, cleavage of the oligonucleotide,for example, causes emission of a fluorescent signal.

DETAILED DESCRIPTION

[0048] The invention provides methods for studying interactions betweenmolecules in vitro or in vivo. In one group of embodiments, the methodsare useful for determining the positions of a protein on either DNA orRNA. These methods are referred to generally as “PINPOINT” methods. Inother embodiments, the invention provides methods of detectinginteractions between two molecules with high resolution and sensitivity.These methods, are referred to generally as “FLASHPOINT” methods.

[0049] PINPOINT Methods

[0050] In pinpoint methods, a target nucleic acid binding site iscleaved in a cell with a chimeric guide-endonuclease fusion molecule. Ingeneral, this involves providing a cell having the target nucleic acidand a chimeric guide molecule-endonuclease fusion molecule andpermitting the fusion molecule to cleave the target nucleic acid,thereby producing a cleaved target nucleic acid with a site of cleavage.The target nucleic acid can be native to the cell, or introduced intothe cell, e.g., by cellular transduction with the target nucleic acid.The chimeric guide endonuclease molecule is provided by transducing thecell with a nucleic acid encoding the molecule and expressing themolecule, or by delivering the molecule directly to the cell, i.e., byreceptor-mediated uptake of the molecule, by liposomal delivery, or thelike.

[0051] Uses of PINPOINT Methods

[0052] PINPOINT methods are useful for a number of different purposes.PINPOINT methods are used to identify genes involved in any diseasestate, including, inter alia, cancer, infection, Alzheimer's disease,atherosclerosis, AIDS and the like. PINPOINT methods are also suitablefor studying basic questions regarding gene regulation, cell cycleregulation, DNA replication and DNA rearrangement. As described herein,methods are provided for monitoring nucleic acid-protein interactions invivo.

[0053] A brief outline of some of the applications for PINPOINT methodsis provided below; one of skill will immediately recognize many otheruses for PINPOINT methods upon viewing this disclosure.

[0054] 1. Pinpoint methods provide basic laboratory research tools. Manyimportant events in the life of a cell or an organism occur on or nearthe DNA and RNA: transcription, DNA replication, repair, DNAmodifications such as methylation, DNA rearrangement, attachment orpositioning relative to nuclear structures such as the nuclear matrix orlamin, posttranscriptional modification of RNA, transport of RNA,attachment or positioning relative to cellular structures, sequestrationand translation and cell cycle regulation. PINPOINT methods are appliedto study all of these events in vivo. Although a number ofprotein-nucleic acids interactions have been studied in vitro, this doesnot accurately duplicate the environment that exists in a living celland therefore misleads or leaves out important molecular events thatoccur in vivo. Furthermore, interactions of proteins not directly boundto DNA or RNA but positioned near DNA or RNA through protein-proteininteraction can be studied. In addition, PINPOINT methods allow thevisualization of protein complex nucleic acid interactions that aredifficult to study where there are insufficient amounts of the purifiedprotein complex. With PINPOINT, a pointer is incorporated into thecomplex and positioned by the cellular machinery on DNA or RNA, removingthe need for protein purification. We found that electroshocking cellsin the presence of calcium activated expressed chimeric proteinscomprising MNase activity without killing cells. It is possible tocleave a particular DNA or RNA sequence by targeting a pointer havingMNase activity to that site and activating MNase with calcium.

[0055] 2. Pinpoint methods provide basic medical research tools. Anumber of diseases result from defective regulation of gene expressioneither at the transcriptional or translational level. Genes for proteinsplaying important roles in these diseases have been cloned but theirtargets on DNA or RNA are not completely known. For example, only a fewgenes directly regulated by the tumor suppressor protein p53 are known.By using PINPOINT methods, the remaining targets (and regulators) of p53can be determined. By knowing the position of p53 in the genome, thefunctions of p53 can be determined. Understanding how p53 suppressestumor growth is an important step in developing treatments for cancer.In a similar way, PINPOINT methods enhance the understanding of,diagnosis of and treatment of many diseases.

[0056] 3. Genome project. In several years, all of the human genome willbe sequenced. The yeast Saccharomyces cerevisiae genome has beencompletely sequenced. Using point directory methods described herein, amap of all genomic positions for a protein of interest can be createdfor any given moment during the life cycle of a cell. Such genome widepictures of protein-DNA/RNA interaction or protein-protein interactionwill reveal important information on gene expression and cellularstructures, including active promoter positions throughout the genome.In humans, knowledge of a DNA sequence for a gene by itself cannotidentify the site of a promoter for the gene. However, by determiningthe genomic positions of protein bound to the promoter of alltranscribed genes, one can determine the positions of all the activepromoters in a given cell. In addition to the tools provided for basicresearch, the ability to accurately monitor promoter activity for manygenes simultaneously aids in diagnosis of any disease state associatedwith gene misregulation, including all forms of cancer.

[0057] 4. Clinical tools. The repertoire of genes being transcribed ortranslated at any given moment reflects the diseased state of anabnormal cell. By determining the positions of RNA polymerase in thecell, one can determine what genes are transcribed in the cell; bydetermining the positions of the translation proteins such as ribosomesor EIF-4, one can determine what RNAs are being translated. Misplacementof a protein in the genome can lead a cell to a diseased state;therefore, determining genomic positions of crucial proteins can alsoaid in diagnostic methods.

[0058] 5. Drug Screening Assays. Because the effects of potentialmodulators on protein-nucleic acids can be measured, a variety of verypowerful screening assays for drugs are provided. The ability to screenrapidly for agents which modulate direct or indirect protein-nucleicacid interactions is of immediate value to the pharmaceutical industry.

[0059] Endonuclease Molecules

[0060] Micrococcal endonuclease produces a 3′ phosphate and a 5′hydroxyl at the site of cleavage. Thus, guide fusion proteins of theinvention which produce this activity are provided. Other guide fusions,such as Fok I (or other class IIS restriction endonuclease) fusions,produce a 5′ phosphate and a 3′ hydroxyl at the cleavage site. In eithercase, the cleaved target is treated in any of a variety of ways tofacilitate amplification and/or detection of the target nucleic acid.Commonly, oligonucleotides are ligated to the cleaved target nucleicacid, or a terminal transferase enzyme is used to provide the cleavedtarget with defined ends for purification, subsequent PCR amplificationand/or cloning.

[0061] In one embodiment, a first double-stranded target DNA and,optionally, a second double-stranded target DNA which correspond to eachside of the cleavage site are made. A variety of techniques can beutilized to make the double-stranded nucleic acid, including oligoligation, terminal transferase extension, enzymatic cleavage orligation, PCR using primers to known sequences in the vicinity of thetarget, and the like. In one embodiment, a purification oligonucleotidecomprising a class IIS restriction site is ligated to the first and/orsecond double-stranded target DNA, thereby producing a ligated targetDNA. The purification oligonucleotide optionally further includessequences for subsequent purification, cloning or PCR reactions, such asa class I restriction site, a region which hybridizes to a PCR primerand/or to a sequencing primer, a restriction site for cloning, adetectable label (e.g., biotin or avidin), or the like. The doublestranded ligated target DNAs are cleaved with the class IIS restrictionenzymes, producing target DNAs with termini that can be used insubsequent purification, cloning, or the like. The target DNAs withtermini are isolated, typically by capture of the oligonucleotides whichwere ligated to the target DNAs. In one embodiment, the captured targetDNAs are released by cleavage with a restriction enzyme which recognizesa site in the first and, optionally, the second ligated target DNAs,optionally by capture of the first and second detectable label andcleavage of the captured label with the first and second class Irestriction enzyme. Optionally, the ligated target DNAs are ligated toform a ligated target site, which is optionally concatemerized forsubsequent cloning and sequencing.

[0062] In embodiments in which a 3′ phosphate is produced in cleavage bythe guide-endonuclease fusion molecule, one of a variety of techniquesare used for detection, isolation and/or amplification of the targetnucleic acid. Typically, the 3′ phosphate is dephosphorylated at thesite of cleavage, thereby producing a 3′ dephosphorylated cleavage end.This cleavage end is extended (e.g., by oligo ligation, or terminaltransferase extension) and the 3′ end is typically amplified, e.g., byPCR.

[0063] In certain embodiments, the target nucleic acid is a plasmid. Inone class of embodiments, where the endonuclease domain of the chimericmolecule is a calcium inducible fusion partner such as micrococcalnuclease, the cell is permeabilized and treated with calcium to inducethe calcium inducible chimeric molecule, which cleaves a single strandof the plasmid, leaving a 3′ phosphate and a 5′ hydroxyl at the site ofcleavage. Plasmid nucleic acids are isolated from the cell, includingthe cleaved plasmid, thereby producing isolated plasmid nucleic acids.The cleaved plasmid is primer extended using a primer extension primerwhich is complementary to a strand of the plasmid comprising thecleavage site, thereby producing a double-stranded blunt end at the siteof cleavage. A trapping oligonucleotide is ligated to thedouble-stranded blunt end, and the cleaved plasmid is PCR amplifiedusing a PCR reaction mixture comprising an oligonucleotide whichhybridizes to the trapping oligonucleotide, and, optionally, the primerextension primer, thereby amplifying the nucleic acid.

[0064] The 3′ end can be dephosphorylated, thereby producing a 3′dephosphorylated cleavage end, which can be extended, e.g., by ligatingan oligonucleotide to the end, or by treating the end with a terminaltransferase enzyme. In many embodiments, the resulting extended 3′ endis amplified and/or detected. Optionally, an internal PCR primer is usedto prime PCR for amplification of the extended end.

[0065] In certain embodiments, cleavage of the target nucleic acid bythe guide molecule-endonuclease chimera produces a 3′ phosphate and a 5′hydroxyl at the site of cleavage. Typical processing steps performed onthe cleaved nucleic acid include: 5′ phosphorylating the cleaved nucleicacid at the site of cleavage to produce a 5′ phosphorylated site; 3′dephosphorylating the cleaved nucleic acid at the site of cleavage;extending the 3′ end of the cleavage site with a terminal transferaseenzyme; extending the 3′ end of the cleavage site by ligating a 3′extension oligonucleotide to the cleaved nucleic acid; primer extendingthe cleaved target nucleic acid using a primer extension primer which iscomplementary to a strand of the nucleic acid comprising the cleavagesite, thereby producing a double-stranded blunt end at the site ofcleavage; extending the 5′ end by ligating a 5′ extensionoligonucleotide to the cleaved target DNA; PCR amplifying target nucleicacid using a primer complementary to the 5′ extension oligonucleotide;and, performing nested PCR on amplified nucleic acids. Any or all ofthese steps are optionally practiced in the methods of the invention.

[0066] In one class of embodiments, the target nucleic acid is a genomicDNA and the chimeric molecule is calcium inducible. Optionally in thisclass, a cell is co-transfected with a marker vector and a chimericnucleic acid vector encoding the chimeric guide molecule. The cell iscultured under conditions which permit expression of a marker encoded bythe marker vector and the chimeric guide molecule, thereby producing amarked cell which expresses the marker and the chimeric guide molecule.The marked cell is typically isolated (i.e., by tracking the marker),thereby providing an isolated cell. The isolated cell is permeabilizedwith a mild detergent and treated with calcium, thereby inducing thecalcium inducible chimeric molecule, which cleaves at least one strandof the target nucleic acid.

[0067] Optionally, where the chimeric molecule leaves a 3′ phosphate anda 5′ hydroxyl at the site of cleavage on the target nucleic acid, the 5′hydroxyl is phosphorylated, providing a 5′ phosphate site on the cleavednucleic acid for primer extension, oligo ligation, or other processingsteps which aid in amplifying, purifying, cloning or detecting thecleaved target. For example, a trapping oligonucleotide is ligated tothe 5′ phosphate site to produce a target-linker nucleic acid. Thistrapping oligonucleotide is used as a molecular tag to amplify and/orpurify the target-linker nucleic acid. Optionally, the site of cleavageis also or separately 3′ dephosphorylated at the site of cleavage. Thisis performed separate from or in parallel with the 5′ phosphorylation ofthe 5′ hydroxyl. The 3′ end is extended using a terminal transferaseenzyme to produce an extended-target nucleic acid, or the 3′ end isextended by ligation of an oligonucleotide. In either case, theextended-target nucleic acid is optionally PCR amplified to produce anamplified target-linker nucleic acid. Optionally, the 3′ end of theresulting amplified target nucleic acid is extended using oligo ligationor terminal transferase, and again PCR amplified or detected.

[0068] In one embodiment, the cleavage site is detected by PCR. Forexample, PCR primers bracketing the cleavage site are used to prime aPCR reaction. If the target site is cleaved, the nucleic acid is notexponentially amplified by PCR. In a variation, determining thepercentage cleaved is performed by this method using standardquantitative PCR methods. See, PCR Protocols A Guide to Methods andApplications (Innis et al. eds) Academic Press Inc. San Diego, Calif.(1990) (Innis).

[0069] In one class of embodiments, the target nucleic acid is an RNA.In certain embodiments, cleavage of the RNA by the guidemolecule-endonuclease chimera produces a 3′ phosphate and a 5′ hydroxylat the site of cleavage. Typically, RNA is isolated from the cell,thereby producing isolated RNA. Optionally, an RNA terminator is coupledto 3′ hydroxyls present in the isolated RNA, to block subsequentamplification of RNAs other than the target RNA. Typically, the 3′phosphate is dephosphorylated with a kinase enzyme or a phosphataseenzyme to produce a dephosphorylated target RNA. Optionally, anoligonucleotide is ligated to the dephosphorylated target RNA. In oneembodiment, the oligonucleotide comprises a binding site for a class IISrestriction enzyme, which can be used in subsequent purification steps.

[0070] Typically, the cleaved target RNA is reverse transcribed forfurther processing, optionally using a reverse transcription primerwhich hybridizes to an oligonucleotide ligated to the RNA to prime thereverse transcription reaction, thereby producing a reverse transcribednucleic acid. Generally, the resulting reverse transcribed RNA-DNAhybrid nucleic acid is treated with an enzyme with RNAse H activity, anda second enzyme with DNA polymerase activity to convert the hybrid intoa double-stranded target DNA.

[0071] The double-stranded target DNA can be cleaved with a restrictionenzyme, thereby producing a restricted target DNA. Ligation of anoligonucleotide to the restricted target DNA facilitates furtherpurification, amplification, cloning or sequencing of the restrictedtarget DNA.

[0072] Typical processing steps for treating cleaved target nucleicacids of the invention can include one or more of: random primerextending the cleaved target nucleic acid, ligating a blockingoligonucleotide to the cleaved target nucleic acid 5′ phosphorylatingthe cleaved target nucleic acid, cleaving the target nucleic acid with arestriction enzyme, cleaving an amplified target nucleic acid with arestriction enzyme, trapping cleaved target DNA to produce trappedtarget DNA, ligating a reaching oligonucleotide to the trapped targetDNA, amplifying the target DNA using PCR, blocking a 3′OH terminal ofthe cleaved target nucleic acid with a terminal transferase, blocking a3′OH terminal of the cleaved target nucleic acid with a blockingoligonucleotide, 3′ dephosphorylating the cleaved target nucleic acid,3′ end extending the cleaved target nucleic acid with a terminaltransferase enzyme, 3′ end extending the cleaved target nucleic acid byligating an oligonucleotide onto the 3′ end of a cleaved target nucleicacid, primer extending the cleaved target nucleic acid, cloning theguide molecule-chimera, or a combination of any of these steps.

[0073] Transducing Cells and Detecting Cleavage Products for PINPOINTMethods

[0074] In one class of embodiments, methods of cleaving a target nucleicacid binding site in a cell with a chimeric guide-endonuclease fusionmolecule are provided. Typically, a cell having the target nucleic acidand a chimeric nucleic acid encoding the chimeric guidemolecule-endonuclease fusion molecule is provided, the nucleic acids areexpressed and the resulting fusion molecule cleaves the target nucleicacid.

[0075] In certain embodiments, the guide molecule-chimera is cloned andexpressed in the cell. In other embodiments, the fusion molecule isdelivered to the cell (e.g., by liposome delivery, receptor mediateduptake or the like). Optionally, the cell is provided by transducing thecell with either the target nucleic acid or the guide-endonuclease, orboth. The cell optionally comprises further components such as areporter gene, a selectable marker, a label, specific genomic mutations,or the like.

[0076] The cleaved target nucleic acid is optionally amplified, and/ordetected. The cleaved target is optionally cloned, and/or sequenced, andis optionally subjected to one of a variety of further processing steps,as described herein. Detection methods include nested PCR, Southernblotting, northern blotting, and cloning and sequencing the targetnucleic acid.

[0077] Transduction of cells with a target nucleic acids, nucleic acidsencoding a chimeric guide-endonuclease fusion molecule, marker genes, orthe like, is performed using standard methods. Host cells are competentor rendered competent for transformation by various means. There areseveral well-known methods of introducing DNA or other materials (e.g.,chimeric guide-endonuclease fusion molecules) into cells. These includecalcium phosphate precipitation, fusion of the recipient cells withbacterial protoplasts containing the DNA, treatment of the recipientcells with liposomes containing the DNA, DEAE dextran, receptor-mediatedendocytosis, electroporation and micro-injection of the DNA directlyinto the cells. Electroporation is a preferred method when a nucleicacid is to be transduced into the cell. Examples of appropriate cloningand transduction techniques, as well as related techniques directed todetecting and assessing nucleic acids by sequencing, Southern analysis,northern analysis and the like are found in Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology volume 152 AcademicPress, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989)Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook); and CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (1996 Supplement) (Ausubel).

[0078] As discussed, receptor-mediated endocytosis provides an efficientmeans of causing a cell to ingest material which binds to a cell surfacereceptor. See, Wu and Wu (1987) J. Biol. Chem. 262:4429-4432; Wagner etal. (1990) Proc. Natl. Acad. Sci. USA 87:3410-3414, and EP-A1 0388 758.To cause a cell to ingest a nucleic acid or a chimeric molecule, thechimeric molecule or nucleic acid is complexed to material recognized bya cellular receptor. For example, naked plasmid DNA boundelectrostatically to poly-l-lysine or poly-l-lysine-transferrin which islinked to defective adenovirus mutants can be delivered to cells withtransfection efficiencies approaching 90% (Curiel et al. (1991) ProcNatl Acad Sci USA 88:8850-8854; Cotten et al. (1992) Proc Natl Acad SciUSA 89:6094-6098; Curiel et al. (1992) Hum Gene Ther 3:147-154; Wagneret al. (1992) Proc Natl Acad Sci USA 89:6099-6103; Michael et al. (1993)J Biol Chem 268:6866-6869; Curiel et al. (1992) Am J Respir Cell MolBiol 6:247-252, and Harris et al. (1993) Am J Respir Cell Mol Biol9:441-447). The adenovirus-poly-l-lysine-DNA conjugate binds to thenormal adenovirus receptor and is subsequently internalized byreceptor-mediated endocytosis.

[0079] Suitable methods for the detection of cleaved target nucleicacids in biological samples include Southern analysis, PCR, northernanalysis, in situ hybridization (including fluorescent in situhybridization (FISH), reverse chromosome painting, FISH on DAPI stainedchromosomes, generation of Alphoid DNA probes for FISH using PCR, PRINSlabeling of DNA, free chromatin mapping and a variety of othertechniques described in Tijssen (1993) Laboratory Techniques inbiochemistry and molecular biology—hybridization with nucleic acidprobes parts I and II, Elsevier, New York, and, Choo (ed) (1994) MethodsIn Molecular Biology Volume 33—In Situ Hybridization Protocols, HumanaPress Inc., New Jersey (see also, other books in the Methods inMolecular Biology series)). PCR is a preferred amplification anddetection method for amplifying and detecting cleaved target nucleicacids.

[0080] A variety of automated solid-phase detection techniques are alsoappropriate. For instance, very large scale immobilized polymer arrays(VLSIPS™), available from Affymetrix, Inc. in Santa Clara, Calif. areused for the detection of nucleic acids. See, Tijssen (supra.), Fodor etal. (1991) Science, 251: 767-777; Sheldon et al. (1993) ClinicalChemistry 39(4): 718-719, and Kozal et al. (1996) Nature Medicine 2(7):753-759. In one embodiment, the invention provides methods of detectingtarget nucleic acids, in which target nucleic acids, or amplifiednucleic acids corresponding to target nucleic acids, are hybridized toan array of nucleic acids. For example, in the pinpoint address methodsdescribed supra, oligonucleotides which hybridize to all possiblePINPOINT address sequences are optionally synthesized on a DNA chip(such chips are available from Affymetrix) and the PINPOINT addressfragments are hybridized to the chip for simultaneous analysis ofmultiple target nucleic acids, or multiple amplified target nucleicacids. The target nucleic acids that are present in the sample which isassayed are detected at specific positions on the chip.

[0081] Methods of culturing cells transduced with target nucleic acids,nucleic acids encoding chimeric guide-endonuclease fusion proteins,marker nucleic acids and the like are known, and are taught in Berger,Sambrook and Ausubel, all supra, as well as Freshney (Culture of AnimalCells, a Manual of Basic Technique, third edition Wiley-Liss, New York(1994)) and the references cited therein. See, also, Kuchler et al.(1977) Biochemical Methods in Cell Culture and Virology, Kuchler, R. J.,Dowden, Hutchinson and Ross, Inc. PINPOINT methods are applied toessentially any cell system available, including bacterial cells, insectcells, plant cells, yeast cells, fungal cells and the like by expressingor delivering the chimeric guide endonuclease molecule to the cell.Similarly, a transgenic organism expressing the chimeric guideendonuclease molecule can be made using available techniques. Techniquesfor making transgenic plants, insects and vertebrates (includingmammals) are well known and can be applied to the present invention.

[0082] Modification of the Cleaved Target Nucleic Acid

[0083] In some embodiments, cleavage of the target nucleic acid by thechimeric guide-endonuclease fusion molecule leaves a 5′ P and a 3′ OH atthe site of cleavage. In these embodiments, the ends are optionallytreated by ligation of an oligonucleotide, or extension by a terminaltransferase to leave a molecular “tag” to which subsequent PCR primeroligonucleotides and/or detection oligonucleotides are hybridized insubsequent amplification and/or detection strategies.

[0084] In other embodiments, cleavage by the chimeric guide-endonucleasefusion molecule leaves a 3′ P and a 5′ OH terminus. For example,cleavage by a micrococcal endonuclease domain leaves 3′ P and a 5′ OHtermini at the cleavage site. Targets with these termini are amplifiedand/or isolated by taking advantage of these unique termini. Forexample, background in subsequent amplification steps is reduced byligating a blocking reagent such as an oligonucleotide oroligonucleotide analogue onto all available termini in a samplefollowing the cleavage reaction. A ligase enzymes which does not ligatean oligonucleotide to the 3′ P or the 5′ OH is used, resulting in all ofthe termini which are unrelated to the target cleavage site being markedby the blocking reagent which blocks subsequent ligation to theunrelated termini. For example, in RNA PINPOINT strategies where 3′ Pand 5′ OH ends are made with the chimeric molecule, any regular 3′ OHends in the RNA mixture are first blocked with T4 RNA ligase and aterminating ribose nucleotide such as a 3′-O-methylguanosine 5′triphosphate. This prevents unwanted ligation of oligonucleotides to theRNA in subsequent amplification, purification or reverse transcriptionprotocols.

[0085] Subsequently, the sample is treated with a kinase or phosphataseenzyme as appropriate to convert the 5′ OH to a 5′ P, and the 3′ P to a3′ OH. The cleavage sites then have an oligonucleotide ligated whichpermits subsequent processing. Ligase, phosphatase, kinase and terminaltransferase enzymes are widely available from a variety of commercialsources known to one of skill. Sambrook, Ausubel, Berger and Innis, allsupra, provide appropriate reaction conditions for these enzymes, as docommercial suppliers of the enzymes.

[0086] Oligonucleotide Ligation to Reaction Cleavage Products

[0087] In the methods of the invention, a chimeric guide-endonucleasefusion molecule is used to cleave a target nucleic acid. The cleavagesite produced by this cleavage reaction provide cleavage ends which areconveniently used for attaching molecular tags such as oligonucleotides,which are used in subsequent steps for purification, amplificationand/or detection of the target nucleic acid.

[0088] Oligonucleotides are typically synthesized chemically accordingto the solid phase phosphoramidite triester method described by Beaucageand Caruthers (1981), Tetrahedron Letts., 22(20):1859-1862, e.g., usingan automated synthesizer, as described in Needham-VanDevanter et al.(1984) Nucleic Acids Res., 12:6159-6168. Oligonucleotides can also becustom made and/or ordered from a variety of commercial sources known topersons of skill, or produced recombinantly. Purification ofoligonucleotides, where necessary, is optionally performed by nativeacrylamide gel electrophoresis or by anion-exchange HPLC as described inPearson and Regnier (1983) J. Chrom. 255:137-149, or by variouschromatographic methods known to persons of skill. A variety ofspecialized devices are commercially available for oligonucleotidepurification. The sequence of oligonucleotides can be verified using thechemical degradation method of Maxam and Gilbert (1980) in Grossman andMoldave (eds.) Academic Press, New York, Methods in Enzymology65:499-560. Ligation of oligonucleotides to a cleavage site is performedusing a ligase enzyme, as described in Sambrook, Berger and Ausubel, allsupra, or chemically ligated using standard organic coupling reactionsas described in March (Advanced Organic Chemistry Reactions, Mechanismsand Structure 4th ed J. Wiley and Sons (New York, 1992), and thereferences cited therein.

[0089] In an alternative embodiment, a terminal transferase enzyme isused to synthesize a chain of nucleotides on a cleavage site. Typically,a nucleotide with a selected polynucleotide sequence is produced, suchas a poly-G sequence. This added sequence can act as a molecular tag forsubsequent processing steps in the same way as a ligatedoligonucleotide.

[0090] As described, in certain embodiments, unusual cleavage ends areproduced, leaving a 3′ phosphate and a 5′ OH. To make these endssuitable for ligation or terminal transferase addition, the site istreated with a kinase (e.g., T₄ kinase) or phosphatase (calf alkalinephosphatase) enzyme to add or delete a phosphate from the site asneeded. Where ligation of an oligonucleotide to an RNA is desired, a T₄RNA ligase or similar enzyme can be used to ligate the oligonucleotideto the RNA.

[0091] In certain embodiments, an oligonucleotide or nucleic acidcorresponding to the target nucleic acid comprises a detectable label.Detectable labels are compositions detectable by spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Forexample, useful labels include ³²P, ³³P, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, dioxigenin, or haptens and proteins for which antisera ormonoclonal antibodies are available. In certain embodiments,particularly where the target nucleic acid is a genomic or otherrelatively rare nucleic acid, the label is used in subsequent processingsteps to partially purify a target nucleic acid having anoligonucleotide label. This is typically done by affinity chromatographyusing a cognate ligand to the label, or by isolating the nucleic acid onbeads having a cognate ligand to the label, or the like.

[0092] PCR of Cleaved Target DNA

[0093] A variety of procedures for PCR amplifying cleavedoligonucleotides are provided herein, and other in vitro amplificationmethods are also useful for amplifying a target nucleic acid. In vitroamplification techniques suitable for amplifying sequences to provide alarge nucleic acid or for subsequent analysis, sequencing or subcloningare known. Examples of techniques sufficient to direct persons of skillthrough such in vitro amplification methods, including the polymerasechain reaction (PCR) the ligase chain reaction (LCR), Qβ-replicaseamplification and other RNA polymerase mediated techniques (e.g., NASBA)are found in Berger, Sambrook, and Ausubel, as well as Mullis et al.(1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods andApplications (Innis et al. eds) Academic Press Inc. San Diego, Calif.(1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; TheJournal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl.Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci.USA 87, 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826; Landegren etal., (1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8,291-294; Wu and Wallace, (1989) Gene 4, 560; Barringer et al. (1990)Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology 13: 563-564.Improved methods of cloning in vitro amplified nucleic acids aredescribed in Wallace et al., U.S. Pat. No. 5,426,039. Improved methodsof amplifying large nucleic acids are summarized in Cheng et al. (1994)Nature 369: 684-685 and the references therein. One of skill willappreciate that essentially any RNA can be converted into a doublestranded DNA suitable for restriction digestion, PCR expansion andsequencing using reverse transcriptase and a polymerase. See, Ausubel,Sambrook and Berger, all supra.

[0094] One of skill will appreciate that two single-stranded nucleicacids “hybridize” when they form a double-stranded duplex. The region ofdouble-strandedness can include the full-length of one or both of thesingle-stranded nucleic acids, or all of one single stranded nucleicacid and a subsequence of the other single stranded nucleic acid, or theregion of double-strandedness can include a subsequence of each nucleicacid. An extensive guide to the hybridization of nucleic acids is foundin Tijssen (1993) Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes part I chapter 2“overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York.

[0095] In the context of the present invention, it is common tohybridize a primer to a template nucleic acid for primer extension, orduring PCR. Appropriate solutions and temperatures for hybridization aresequence dependent, with the selection of appropriate hybridizationconditions being routine. See, Tijssen et al., id. Generally, highlystringent hybridization conditions are selected to be about 5-10° C.lower than the thermal melting point (T_(m)) for the specific sequenceat a defined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe. See also, Innis, supra.

[0096] While many sequences can be used to construct primers, selectingoptimal amplification primers is optionally done using computer assistedconsideration of available sequence and excluding potential primerswhich do not have desired hybridization characteristics, and/orincluding potential primers which meet selected hybridizationcharacteristics. This is done by determining all possible nucleic acidprimers, or a subset of all possible primers with selected hybridizationproperties (e.g., those with a selected length and G:C ratio) based uponthe known sequence. The selection of the hybridization properties of theprimer is dependent on the desired hybridization and discriminationproperties primer. In general, the longer the primer, the higher themelting temperature. However, longer primers are not as specific becausea single mismatch has less of a destabilizing effect on hybridizationthan a single mismatch on a short nucleic acid duplex; thus, longprimers can create unwanted PCR products. It is expected that one ofskill is thoroughly familiar with the theory and practice of nucleicacid hybridization and primer selection. Gait, ed. OligonucleotideSynthesis: A Practical Approach, IRL Press, Oxford (1984); W. H. A.Kuijpers Nucleic Acids Research 18(17), 5197 (1994); K. L. Dueholm J.Org. Chem. 59, 5767-5773 (1994); S. Agrawal (ed.) Methods in MolecularBiology, volume 20; and Tijssen (1993) Laboratory Techniques inbiochemistry and molecular biology—hybridization with nucleic acidprobes, e.g., part I chapter 2 “overview of principles of hybridizationand the strategy of nucleic acid probe assays”, Elsevier, New Yorkprovide a basic guide to nucleic acid hybridization. Innis supraprovides an overview of primer selection.

[0097] Most typically, amplification primers are between 8 and 100nucleotides in length, and preferably between about 10 and 30nucleotides in length. Most preferably, the primers are between 15 and45 nucleic acids in length. For example, in one preferred embodiment,the nucleic acid primers are about 17-30 nucleotides in length.

[0098] One of skill will recognize that the 3′ end of an amplificationprimer is more relevant in PCR than the 5′ end. Investigators havereported PCR products where only a few nucleotides at the 3′ end of anamplification primer were complementary to a DNA to be amplified. Inthis regard, nucleotides at the 5′ end of a primer can incorporatestructural features unrelated to the target nucleic acid; for instance,in one preferred embodiment, a second primer hybridization site (or acomplement to such as primer, depending on the application) isincorporated into any amplification primer. For example, anamplification primer derived from a sequencing primer used in a standardsequencing kit, such as one using a biotinylated or dye-labeleduniversal M13 or SP6 primer can be incorporated into an amplificationprimer. Similarly, restriction endonuclease recognition sites areoptionally incorporated in a similar manner.

[0099] In some embodiments, amplification oligonucleotide sequences areselected to hybridize only to a perfectly complementary DNA, with thenearest mismatch hybridization possibility from a DNA sequence unrelatedto the target nucleic acid which is known to exist in a cell having atleast about 50 to 70% hybridization mismatches, and preferably 100%mismatches for the terminal 5 nucleotides at the 3′ end of the primer.

[0100] The amplification primers are optionally selected so that nosecondary structure forms within the primer. Self-complementary primershave poor hybridization properties, because the complementary portionsof the primers self hybridize (i.e., form hairpin structures). Theprimers are also selected so that the primers do not hybridize to eachother, thereby preventing duplex formation of the primers in solution,and possible concatenation and unwanted amplification of the primersduring PCR.

[0101] Where sets of amplification primers (i.e., the 5′ and 3′ primersused for exponential amplification) are of a single length, the primersare optionally selected so that they have roughly the same, andpreferably exactly the same overall base composition (i.e., the same A+Tto G+C ratio of nucleic acids). Where the primers are of differinglengths, the A+T to G+C ratio is determined by selecting a thermalmelting temperature for the primer-DNA hybridization, and selecting anA+T to G+C ratio and probe length for each primer which hasapproximately the selected thermal melting temperature.

[0102] One of skill will recognize that there are a variety of possibleways of performing the above selection steps, and that variations on thesteps are appropriate. Most typically, selection steps are performedusing simple computer programs to perform the selection as outlinedabove; however, all of the steps are optionally performed manually. Oneavailable computer program for primer selection is the Mac Vector™program from Kodak. In addition to commercially available programs forprimer selection, one of skill can easily design simple programs for anyof the preferred amplification steps.

[0103] Screening for in vivo Binding Sites

[0104] The present invention provides methods of screening test nucleicacids for in vivo binding sites which are cleaved by a chimeric guidemolecule. Typically, a cell comprising a chimeric nucleic acid encodingthe chimeric guide molecule and a test nucleic acid is provided, thechimeric nucleic acid is expressed in the cell, thereby producingchimeric guide molecule in the cell and, the cell is incubated underconditions in which the guide molecule is active. If the chimeric guidemolecule cleaves the test nucleic acid, the test nucleic acid comprisesan in vivo binding site for the chimeric guide molecule. The ability totest whether a particular chimeric molecule cleaves a target siteprovides evidence for whether a naturally occurring moleculecorresponding to the guide domain binds the target in vivo. This, inturn, is of value for basic research, for finding targets fortherapeutic genes, for targeting therapeutics to disease-related genes,or the like. For example, if a guide domain corresponding to a tumorsuppressor is found to interact with a target site in the promoter of agene which mediates disease, then therapeutics which modulate theactivity of the tumor suppressor can be tested for an effect on the genewhich mediates disease.

[0105] In one assay of the invention, the test nucleic acid encodes apromoter sequence operably linked to a reporter gene. Detection of thepresence or absence of reporter gene expression is an indicator forwhether the test nucleic acid comprises an in vivo binding site for thechimeric guide molecule. A variety of reporter gene plasmid systems areknown, such as the common chloramphenicol acetyltransferase (CAT) andbeta-galactosidase (e.g., bacterial LacZ gene) reporter systems, thefirefly luciferase gene (See, e.g., Cara et al., (1996) J. Biol. Chem.,271: 5393-5397), the green fluorescence protein (see, e.g., Chalfie etal. (1994) Science 263:802) and many others. Selectable markers whichfacilitate cloning of the vectors of the invention are optionallyincluded. Sambrook and Ausubel, both supra, provide an overview ofselectable markers.

[0106] Promoters selected as targets for testing in conjunction with areporter gene can be from essentially any gene. Preferred promotersdirect expression of pathogen or disease related genes, such as a viralpromoter (HIV-1 or HIV-2 LTRs, HTLV-LTRs, Herpes virus tk promoter, avaccinia promoter, a pox virus promoter, a flu virus promoter, anadenovirus promoter, etc.), an oncogene promoter (e.g., a promoter fromp53, c-myc, fos, etc.), or the like.

[0107] Parallel Screening Formats

[0108] In one class of embodiments, the cell is provided byco-transducing the cell with a plasmid encoding the target nucleic acidand a plasmid encoding the chimeric guide molecule. Optionally, one ormore additional plasmids comprising one or more additional test nucleicacids are also transduced into the cell, and the effect of the chimericguide molecule is assessed simultaneously on more than one test nucleicacid.

[0109] Parallel screening formats are provided, in which a second cellcomprising a second chimeric nucleic acid encoding a second chimericguide molecule and a second test nucleic acid is provided. The secondchimeric nucleic acid is expressed and the effect of the chimericnucleic acid on the second test nucleic acid is monitored. This parallelscreening assay is suitable for automation, providing the ability toscreen the activity of multiple chimeric guide molecules againstmultiple target nucleic acids in a single assay. The use of controlnucleic acids, such as positive control nucleic acids which are known tobe cleaved by a particular guide nucleic acid to verify that thecellular environment permits chimeric molecule activity, and negativecontrol nucleic acids which are known to remain uncleaved in thepresence of the chimeric molecule, are optionally used in the cells ofthe invention.

[0110] A number of cleavage reactions are optionally monitoredsimultaneously, e.g., using a format which permits simultaneous analysisof several samples (microtiter plates, etc.). In a preferred embodiment,the assays are automated, e.g., using robotics for pipetting samplesinto microtiter plates. For example, a Zymate XP (Zymark Corporation;Hopkinton, Mass.) automated robot using Microlab 2200 (Hamilton; Reno,Nev.) pipetting station can be used to transfer cell samples to a 96well microtiter plate to set up several parallel simultaneous assays ofcleavage activity of one or more chimeric molecule on one or more targetnucleic acid.

[0111] The invention provides methods of detecting a nucleic acidbinding molecule modulating agent. In the methods, a cell comprising atest nucleic acid binding site (e.g., a promoter sequence which is boundby a transcription factor) and a chimeric guide-endonuclease molecule isprovided. The cell is contacted with the potential modulating agent, andthe rate of cleavage of the test nucleic acid binding site by thechimeric nucleic acid binding molecule in the presence of the agent inmeasured. Typically, this measurement is compared to the rate ofcleavage of the test nucleic acid in the absence of the modulatingagent. If the cleavage rate is increased or decreased, the potentialmodulator is confirmed to be a modulating agent. To verify that themodulation is due to effects on the guide domain, and not on theendonuclease domain, the modulator is tested for modulatory activityagainst a second chimeric guide molecule which has the same endonucleasedomain as the chimeric molecule first tested. Modulators which do notaffect the activity of second chimeric molecule are specific for theguide domain of the chimeric molecule first tested. Multiple assays arepreferably performed in parallel in the methods of the invention, withseveral agents and/or several activities being screened simultaneously(e.g., in a microtiter format as described), a decided advantage iflarge numbers of agents and/or target nucleic acids and/or chimericmolecules are to be screened.

[0112] Cleavage can be measured in any of a variety of ways, includingquantitative PCR amplification of the cleavage products, assessment of areporter gene construct having a promoter as the target nucleic acids asset forth above, by measuring incorporation of a radioactive nucleotideinto amplification products, or the like. In an embodiment, the amountof product polynucleotide is determined by contacting an amplificationreaction mixture, following a suitable incubation period, with asubstrate which selectively immobilizes or binds to polynucleotides andwhich substantially does not immobilize or bind to mononucleotides orlabelling reagents; one example of such a substrate is a chargedmembrane (e.g., a glass fiber filter such as the 2SC filter fromWhatman, Nylon 66, nitrocellulose, DEAE paper, or the like). This formatis conveniently combined with the microtiter format, e.g., using aunifilter plate (Whatman GF/C glass fiber filter bottom).

[0113] Alternatively, amplification products are chromatographed orelectrophoresed (e.g., PAGE) to separate polynucleotide products fromunincorporated nucleotides or other materials. In either case, productnucleic acids are optionally isolated and cloned, using techniques knownin the art. See, Sambrook, Berger, Ausubel and Innis, all supra.

[0114] In one class of embodiments, the invention provides methods ofcleaving target nucleic acids in vitro or in vivo. In the methods, atarget nucleic acid is contacted by a guide-micrococcal endonucleasefusion molecule in the presence of calcium. The guide-micrococcalendonuclease fusion then cleaves the target nucleic acid. In oneembodiment, the cleavage is performed in situ, e.g., in a tissue or cellsample on a solid substrate such as a microscope slide, an Affymetrixchip, or the like.

[0115] Chimeric Guide-Endonuclease Molecules

[0116] A variety of target nucleic acids are bound by the chimeric guideendonuclease fusion molecules of the invention, including genomicnucleic acids with known sequences of nucleotides, genomic nucleic acidwith unknown sequences of nucleotides, plasmids with sequences of knownnucleotides, plasmids with sequences of unknown nucleotides, RNA withsequences of known nucleotides, and RNA with unknown sequences ofnucleotides. A variety of chimeric guide molecules are used forassessing the target nucleic acids, including those in which the guidedomain is a DNA binding protein, an RNA binding protein, a protein whichbinds to a DNA binding protein, a protein which binds to an RNA bindingprotein, an antibody protein which binds to a DNA binding protein, afirst antibody protein which binds to a second antibody protein, and anantibody protein which binds to an RNA binding protein. In oneembodiment, the target nucleic acid is contacted with a primary antibodywhich binds to a DNA binding protein bound to the target nucleic acid.The chimeric guide-endonuclease fusion protein comprises a secondaryantibody which binds to the primary antibody.

[0117] The guide domain can also be a molecule chemically conjugated tothe endonuclease domain which binds to a target nucleic acid, or to aprotein associated with the target nucleic acid, such as a guide nucleicacid, an antibody fusion or the like. Preferred endonuclease domains areinducible rather than constitutive, conferring the ability to regulatecleavage by the endonuclease domain. A preferred endonuclease ismicrococcal nuclease, which is calcium inducible, and which cleaves bothDNA and RNA. A particularly preferred endonuclease domain is amicrococcal nuclease domain with constitutive in vivo activity which islower than the native micrococcal nuclease enzyme. This lowerconstitutive activity lowers background cleavage activity, increasingthe signal to noise ratio in the methods of the invention.

[0118] Common guide domains include transcription factors (activators),silencers, nuclear receptors, general transcription machinery andmodifiers of these factors, oncogenes (e.g., myc, jun, fos, myb, max,mad, rel, ets, bcl, myb, mos family members etc.), tumor promoters,metastasis and invasiveness promoters or suppressors and theirassociated factors and modifiers; tumor suppressors (e.g. p53, WT1,MDM2, Rb family) and their associated factors and modifiers; DNA repairenzymes and their associated factors and modifiers; DNA rearrangementenzymes and their associated factors and modifiers, cell cycle proteinsand their associated factors and modifiers; chromatin associatedproteins and their modifiers (e.g., kinases, acetylases anddeacetylases); DNA modifying enzymes (e.g., methyltransferases,topoisomerases, helicases, ligases, kinases, phosphatases, polymerases)and their associated factors and modifiers; RNA modifying enzymes andtheir associated factors and modifiers, RNA binding factors (directly orindirectly) and their associated factors and modifiers, factors thatcontrol chromatin, DNA, RNA and RNP (ribonuclear protein) structure,movement and localization and their associated factors and modifiers;factors derived from microbes (e.g., prokaryotes, eukaryotes and virus)and factors that associate with or modify them.

[0119] While a complete protein can be used as a guide domain, portionsof proteins that are capable of binding to nucleic acids, directly orindirectly, are also useful as guide domains. To identify such nucleicacid binding domains, one can perform assays such as an electrophoreticmobility shift assay (EMSA) (Scott et al. (1994) J. Biol. Chem. 269:19848-19858), in which a nucleic acid sequence of interest is allowed toassociate with various fragments of a molecule that is capable ofbinding to the nucleic acid sequence. Association of a portion of theprotein with the nucleic acid will result in a retardation of theelectrophoretic mobility of the nucleic acid. Another method by whichone can identify nucleic acid binding moieties that are suitable for useas guide domains is DNase I footprinting.

[0120] Common polypeptides from which one can obtain a guide domaininclude polypeptides that are involved in transcription, both regulatedand basal transcription. Such polypeptides include transcription factorsand coactivators, silencers, nuclear receptors, general transcriptionmachinery and modifiers of these factors. See, e.g., Goodrich et al.,Cell 84: 825-30 (1996) for a review of proteins and nucleic acidelements involved in transcription. Transcription factors in general arereviewed in Barnes and Adcock, Clin. Exp. Allergy 25 Suppl. 2: 46-9(1995) and Roeder, Methods Enzymol. 273: 165-71 (1996). Databasesdedicated to transcription factors are known. See, e.g., Science269:630. Intracellular receptor transcription factors are described in,for example, Rosen et al., J. Med. Chem. 38: 4855-74 (1995). The C/EBPfamily of transcription factors are reviewed in Wedel et al.,Immunobiology 193: 171-85 (1995). Coactivators and co-repressors thatmediate transcription regulation by nuclear hormone receptors arereviewed in, for example, Meier, Eur. J. Endocrinol. 134(2): 158-9(1996) and Kaiser et al., Trends Biochem. Sci. 21: 342-5 (1996). GATAtranscription factors, which are involved in regulation ofhematopoiesis, are described in, for example, Simon, Nat. Genet. 11:9-11 (1995); Weiss et al., Exp. Hematol. 23: 99-107. TATA box bindingprotein (TBP) and its associated TAF polypeptides (which include TAF30,TAF55, TAF80, TAF110, TAF150, and TAF250) are described in Goodrich andTjian, Curr. Opin. Cell Biol. 6: 403-9 (1994) and Hurley, Curr. Opin.Struct. Biol. 6: 69-75 (1996). The STAT family of transcription factorsare reviewed in, for example, Barahmand-pour et al., Curr. Top.Microbiol. Immunol. 211: 121-8 (1996). Transcription factors involved indisease are reviewed in Aso et al., J. Clin. Invest. 97: 1561-9 (1996).

[0121] Kinases, phosphatases, and other proteins that modifypolypeptides involved in gene regulation are also useful as guidedomains. Such modifiers are often involved in switching on or offtranscription mediated by, for example, hormones. Kinases involved intranscription regulation are reviewed in Davis, Mol. Reprod. Dev. 42:459-67 (1995), Jackson et al., Adv. Second Messenger Phosphoprotein Res.28: 279-86 (1993), and Boulikas, Crit. Rev. Eukaryot. Gene Expr. 5: 1-77(1995), while phosphatases are reviewed in, for example, Schonthal,Semin. Cancer Biol. 6: 239-48 (1995). Nuclear tyrosine kinases aredescribed in Wang, Trends Biochem. Sci. 19: 373-6 (1994).

[0122] As described, guide domains can also be obtained from the geneproducts of oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets,bcl, myb, mos family members, etc.) and tumor suppressors (e.g., p53,WT1, MDM2, Rb family, and the like) and their associated factors andmodifiers. Oncogenes are described in, for example, Cooper, Oncogenes,2nd ed., The Jones and Bartlett Series in Biology, Boston, Mass., Jonesand Bartlett Publishers, 1995. The ets transcription factors arereviewed in Waslylk et al., Eur. J. Biochem. 211: 7-18 (1993) andCrepieux et al., Crit. Rev. Oncog. 5: 615-38 (1994). myc oncogenes arereviewed in, for example, Ryan et al., Biochem. J. 314: 713-21 (1996).The jun and fos transcription factors are described in, for example, TheFos and Jun Families of Transcription Factors, Angel P E, Herrlich P A,eds. Boca Raton, Fla., CRC Press, 1994. The max oncogene is reviewed inHurlin et al., Cold Spring Harb. Symp. Quant. Biol. 59: 109-16. The mybgene family is reviewed in Kanei-Ishii et al., Curr. Top. Microbiol.Immunol. 211: 89-98 (1996). The mos family is reviewed in Yew et al.,Curr. Opin. Genet. Dev. 3: 19-25 (1993).

[0123] Tumor promoters, metastasis and invasiveness promoters orsuppressors and their associated factors and modifiers are also suitablefor use as guide domains. Proteins involved in carcinogenesis, includingtumor suppressors and activators, are reviewed in Schmandt et al., Clin.Chem. 39 (11 Pt 2): 2375-85 (1993) and Kelley et al., Adv. Intern. Med.39: 93-122 (1994). Tumor suppressors are reviewed in Hinds et al., Curr.Opin. Genet. Dev. 4: 135-41 (1994). The p53 tumor suppressor inparticular is described in Hainaut, Curr. Opin. Oncol. 7: 76-82 (1995)and Cox and Lane, Bioessays, 17: 501-8 (1995), while the Rb family isreviewed in Sidle et al., Crit. Rev. Biochem. Mol. Biol. 31: 237-71(1996). Invasiveness promoters and suppressors are reviewed in, forexample, Mareel et al., Mol. Biol. Rep. 19: 45-67 (1994).

[0124] The chimeric endonucleases can also include a guide domainpolypeptide that is obtained from DNA repair enzymes and theirassociated factors and modifiers. DNA repair systems are reviewed in,for example, Vos, Curr. Opin. Cell Biol. 4: 385-95 (1992); Sancar, Ann.Rev. Genet. 29: 69-105 (1995); Lehmann, Genet. Eng. 17: 1-19 (1995); andWood, Ann. Rev. Biochem. 65: 135-67 (1996). DNA rearrangement enzymesand their associated factors and modifiers (see, e.g., Gangloff et al.,Experientia 50: 261-9 (1994); Sadowski, FASEB J. 7: 760-7 (1993)), cellcycle proteins and their associated factors and modifiers are alsouseful as guide domains. For example, proteins involved in DNAreplication can be used to construct chimeric endonucleases. DNAreplication proteins are described in Kearsey et al., Curr. Opin. Genet.Dev. 6: 208-14 (1996) and Donovan et al., Curr. Opin. Genet. Dev. 6:203-7 (1996). Cell cycle proteins are also described in Stein et al.,Int. J. Obes. Relat. Metab. Disord. 20 Suppl 3: S84-90 (1996).

[0125] Similarly, guide domain polypeptides can be derived from DNAmodifying enzymes (e.g., methyltransferases, topoisomerases, helicases,ligases, kinases, phosphatases, polymerases) and their associatedfactors and modifiers. Helicases are reviewed in Matson et al.,Bioessays, 16: 13-22 (1994), and methyltransferases are described inCheng, Curr. Opin. Struct. Biol. 5: 4-10 (1995). Chromatin associatedproteins and their modifiers (e.g., kinases, acetylases anddeacetylases), such as histone deacylase (Wolffe, Science 272: 371-2(1996)) are also useful as guide domains.

[0126] RNA modifying enzymes and their associated factors and modifiers,RNA binding factors (directly or indirectly) and their associatedfactors and modifiers are also useful as guide domains. For review ofprotein-RNA interactions, see, Draper, Ann. Rev. Biochem. 64: 593-620(1995) and Burd et al., Science 29: 615-21 (1994). RNP domains arereviewed in Nagai et al., Trends Biochem. Sci. 20: 235-40 (1995).

[0127] Factors that control chromatin, DNA, RNA and RNP (ribonuclearprotein) structure, movement and localization and their associatedfactors and modifiers; factors derived from microbes (e.g., prokaryotes,eukaryotes and virus) and factors that associate with or modify them canalso be used to obtain guide domains.

[0128] Guide domains can bind to a target nucleic acid directly, orindirectly such as by binding to a protein that is itself directly orindirectly associated with the target nucleic acid. Antibodies thatspecifically bind to proteins that can become directly or indirectlyassociated with a target nucleic acid are one example of a guide domainthat indirectly binds to a target nucleic acid.

[0129] Common endonuclease domains include the cleavage domain of arestriction endonuclease which has a cleavage domain separate from therecognition domain (HphI, MboII, BbvI, FokI, HgaI, SfaNI, BspMI),activatable endonucleases (Micrococcal endonucleases), and nucleic acidswith cleavage activity (ribozymes and the like).

[0130] Linker domains are typically polypeptide sequences, such as polygly sequences of between about 5 and 200 amino acids. In someembodiments, proline residues are incorporated into the linker toprevent the formation of significant secondary structural elements bythe linker. Preferred linkers are often flexible amino acid subsequenceswhich are synthesized as part of a recombinant fusion protein. In oneembodiment, the flexible linker is an amino acid subsequence comprisinga proline such as Gly(x)-Pro-Gly(x) where x is a number between about 3and about 100. In other embodiments, a chemical linker is used toconnect synthetically or recombinantly produced guide and endonucleasesubsequences. Such flexible linkers are known to persons of skill in theart. For example, poly(ethelyne glycol) linkers are available fromShearwater Polymers, Inc. Huntsville, Ala. These linkers optionally haveamide linkages, sulfhydryl linkages, or heterofinctional linkages.

[0131] As described, recombinant guide endonuclease fusion proteins ofthe invention are preferably made via recombinant ligation of nucleicacids encoding the constituent parts of the fusion protein (e.g., guide,linker and endonuclease) and expression of the resulting construct.Instructions sufficient to direct one of skill through such cloningexercises are found in Sambrook, Berger, Ausubel and Innis, all supra.Generating a guide, linker or endonuclease domain from a known protein,linker amino acid, or endonuclease protein is easily performed by one ofskill. Clones for many suitable components are publicly available. Wheresuch clones are not easily obtained from public sources, they are easilymade by PCR amplifying the nucleic acid from a biological sample such asa cell, or a genomic or cDNA library, i.e., by designing PCR primers tocomplement the known nucleic acid sequences (procedures for selectingPCR primers are described supra). Sequences for guide, endonuclease orlinker domains, including those exemplified above, are easily found bysearching public repositories of nucleic acid sequence. Well-establishedrepositories of sequence information include GenBank™, EMBL, DDBJ andthe NCBI, and there are many other databases which are also known. PCRamplified nucleic acids are optionally subcloned to facilitatesubsequent processing, or are used as the source for a nucleic acidencoding a guide, endonuclease or linker domain. Once PCR products, orPCR generated subclones, or publicly available clones are obtained, thecomponents are assembled into a contiguous recombinant nucleic acidencoding the chimeric guide endonuclease fusion protein, and the proteinmade by recombinant expression of the fusion protein.

[0132] One of skill will also recognize that a variety of nucleic acidsequences encode any particular polypeptides due to the codon degeneracypresent in the genetic code. Each of the nucleic acids which encodes agiven polypeptide is described by comparison to the amino acid sequenceof the polypeptide and translation via the genetic code to a codingnucleic acid. In preferred embodiments, a nucleic acids which encodes aparticular polypeptide is optimized for expression is a particular celltype, such as yeast, humans, etc. by reference to sequence codon biastables and substitution of a given sequence with a sequence whichencodes the same polypeptide using codons preferred for the cell type.This typically increases the level of translation of a given nucleicacid, facilitating expression of the encoded chimeric protein.

[0133] Optionally, recombinant components are not synthesizedrecombinantly, but are instead synthesized chemically, e.g., using apeptide synthesizer, or other solid phase protein synthesis technique.See also, March, supra. Solid phase synthesis of polymers, includingbiological polymers is known. See, e.g., Merrifield (1963) J. Am. Chem.Soc. 85: 2149-2154. Solid-phase synthesis techniques have also beenprovided for the synthesis of peptide sequences on, for example, anumber of “pins.” See e.g., Geysen et al. (1987) J. Immun. Meth. 102:259-274. Other solid-phase techniques involve, for example, synthesis ofvarious peptide sequences on cellulose disks supported in a column. See,Frank and Doring (1988) Tetrahedron 44: 6031-6040. Still othersolid-phase techniques are described in U.S. Pat. No. 4,728,502 and WO90/00626. Where one or more domain is not a protein, the domain is fusedchemically to the other domains, i.e., by standard synthetic chemistry,as described supra.

[0134] In one embodiment, the endonuclease is a nucleic acid, which isliked to a guide domain to form the chimeric guide-endonuclease fusionmolecule. For example, a variety of ribozymes are well known. A ribozymeis a catalytic RNA molecule that cleaves other RNA molecules havingparticular nucleic acid sequences. General methods for the constructionof ribozymes, including hairpin ribozymes, hammerhead ribozymes, RNAse Pribozymes (i.e., ribozymes derived from the naturally occurring RNAse Pribozyme from prokaryotes or eukaryotes) are known in the art.Castanotto et al (1994) Advances in Pharmacology 25: 289-317 provides anoverview of ribozymes in general, including group I ribozymes,hammerhead ribozymes, hairpin ribozymes, RNAse P, and axhead ribozymes.In one class of embodiments, the ribozyme RNA recognition domain isdeleted, and the cleavage domain is chemically liked (typically via alinker) to the guide domain.

[0135] In addition, ribonucleoproteins such as the native RNAse Pribonucleoprotein, can be used as endonuclease domains in the guideendonuclease fusion protein of the invention. These ribonucleoproteinsare also typically chemically linked via a linker, to the guide domain.

[0136] Similarly, the guide domain is optionally a nucleic acid whichhybridizes to the target nucleic acid. In this embodiment, the guidedomain is typically chemically linked to the endonuclease domain, usingstandard synthetic methods. An example of an oligonucleotide beingchemically linked to a micrococcal endonuclease protein by chemicalcoupling is found in Corey et al. (1989) Biochemistry 28: 8277-8286.

[0137] Where the linker, guide and endonuclease are all nucleic acids(typically RNA), the nucleic acids are typically produced recombinantly,although they are optionally made by synthetic methods, as described,supra, for oligonucleotides using an automated synthesizer.

[0138] Pinpoint Detection of in vivo Protein-DNA Interactions Using aFok 1 Endonuclease Domain

[0139] In the process of employing PINPOINT to detect protein-DNAinteraction in vivo, a surprising fact was discovered: in vivo, thenuclease domain of Fok 1 predominantly makes single-stranded nicks onthe DNA, rather than double strand cuts as expected from the prior art.This critical observation allowed the development of very sensitivetechniques to detect in vivo protein-DNA interaction; such a developmentwould not have been possible without this discovery.

[0140] In general, these methods utilize ligation or other enzymaticreactions to add an oligonucleotide tag to the site of a Fok 1 nick onthe nucleic acid. This tag, in conjunction with subsequent processingsteps is used to isolate, detect, and/or purify nucleic acid from theregion of the nick.

[0141] Pinpoint Detection of in Vivo Protein-DNA Interactions Using aMicrococcal Endonuclease Domain

[0142] There are drawbacks to the nuclease domain of Fok 1 for in vivoPINPOINT analysis, making chimeric guide-endonuclease fusion proteinswith a Fok 1 domain less preferred. First, the nuclease domain at 25 kdis relatively large, potentially causing stearic problems with the guidedomain, complicating cloning and the like. Second, the nuclease activitycannot easily be regulated; from the moment a chimericguide-endonuclease fusion protein comprising a Fok 1 endonuclease isexpressed in the cell, it is active. As a result, prolonged expressionof the chimeric guide-endonuclease fusion protein (or “pointer”) in acell can be toxic. Lastly, the catalytic rate of Fok 1 nuclease domainis rather low.

[0143] For these reasons, we have also developed PINPOINT methodsemploying micrococcal nuclease chimeric guide-endonuclease fusionproteins in place of Fok 1 fusion proteins. Micrococcal nuclease issmaller in size (16 kd), and therefore causes fewer problems due tointeractions with the guide domain. Unlike Fok 1 nuclease, it requiresmillimolar levels of calcium for maximal activity and therefore it isalmost inactive in normal cells, which have nanomolar levels of calcium.Because micrococcal nuclease is inactive, and therefore non-toxic untilstimulated with calcium, it is possible to create transgenic cells (botheukaryotic and prokaryotic), plants (yeast, algae, monocotyledons,dicotyledons, etc.) and animals (Drosophilla, mice, rats, livestock,etc.) or stable cell lines expressing a chimeric guide-endonucleasefusion protein having a micrococcal endonuclease domain.

[0144] To activate micrococcal nuclease in vivo, the cell membrane ispermeabilized with a mild detergent and incubated in a calciumcontaining buffer for several minutes, resulting in an approximately10,000 fold increase in activity. Another advantage of micrococcalnuclease is that it leaves unique 5′ OH and 3′ P ends after cleavage,facilitating purification strategies designed to target these uniquecleavage products.

[0145] In Vitro Uses for the Chimeric Proteins of the Invention

[0146] In one embodiment, the present invention provides specificchimeric guide-endonuclease proteins having an endonuclease domain.These proteins are useful, inter alia, as custom restriction enzymes forthe cleavage of nucleic acids. Hybrid affinity cleaving proteinscomposed of a DNA binding domain of a protein and a Fok 1 endonucleasehave been used in vitro for cleavage of nucleic acids in a variation ofin vitro footprinting methods. See, Chandrasegaran U.S. Pat. Nos.5,487,994 and 5,436,150; Kim et al. (1996) Proc. Natl. Acad. Sci. USA93:1156-1160 and Kim et al. Proc. Natl. Acad. Sci. USA (1994)91:883-887. One of skill will immediately understand that restrictionenzymes having new recognition and/or cleavage sites are very usefultools in techniques relating to cloning and assessing nucleic acids.

[0147] The present invention provides a new class of hybrid affinitynucleic acid cleavage proteins having a micrococcal nuclease domainlinked via a linker to a guide domain. In addition to the many in vivouses described herein (see also, commonly assigned U.S. Ser. No.08/825,664, filed Apr. 3, 1997 and co-filed U.S. Ser. No.______, filedApr. 2, 1998 as Attorney Docket No. 15280-31820US), these chimericproteins comprising a micrococcal nuclease domain are useful asrestriction enzymes in vitro.

[0148] As restriction enzymes, these chimeric micrococcal endonucleasefusion proteins have several important advantages over the prior art.First, the micrococcal nuclease domain requires calcium for activation,making it possible to regulate the activity of the protein, in vitro orin vivo. For example, cleavage reactions can be stopped by adding acalcium chelator (e.g., EDTA) to the reaction mixture, or started byadding calcium. One of skill will appreciate that partial digestion of aDNA is sometimes necessary during certain cloning procedures. Second,the position of the cleavage site can be selected relative to therecognition site in the chimeric protein by varying the length of thelinker between the micrococcal cleavage domain and the guide domain.Third, micrococcal endonuclease cleaves RNA, giving the chimericproteins of the invention a unique ability to engineer RNA molecules incloning procedures. Fourth, under some reaction conditions micrococcalnuclease cleaves only a single strand of a double-stranded nucleic acid,making it possible to selectively cleave, e.g., a single strand on atarget DNA, leaving ends which can be modified in subsequent reactions.For example, following kinase or phosphatase treatment, an end left bymicrococcal nuclease can be extended using a polymerase, therebyincorporating selected nucleotides into a copy of a target nucleic acid(e.g., radioactive nucleotides used for detecting the copy). Fifth,because the ends made by micrococcal nuclease are unique (leaving a 5′OH and a 3′ P) it is possible to use the unique ends as molecular tagsfor subsequent purification or amplification reactions.

[0149] FLASHPOINT Methods

[0150] The invention also provides methods for detecting whether a firstmolecule is in close proximity to a second molecule. A schematic diagramof the FLASHPOINT methods is shown in FIG. 7. The methods involveattaching a molecular beacon to the first molecule and attaching achimeric endonuclease to the second molecule. To determine whether thefirst molecule is in close proximity to the second molecule, one detectswhether fluorescence is emitted by a fluorophore present on themolecular beacon. Fluorescence emission is indicative of cleavage of themolecular beacon by the endonuclease moiety, thereby causing separationof the fluorophore and a quencher which is also present on the molecularbeacon. Reagents and kits for use in the FLASHPOINT methods aredescribed in more detail in co-filed, commonly assigned U.S. patentapplication Ser. No. ______, filed Apr. 2, 1998 as Attorney Docket No.15280-31820US, which is incorporated herein by reference.

[0151] Uses of FLASHPOINT Methods

[0152] These methods are useful in many types of detection assay whichinvolve detection of intermolecular interactions. Such interactions areinvolved in many biological and chemical events such as, for example,enzymatic reactions, hormone-ligand interactions, drug or toxininteractions with their receptors, and the like. Moieties from which thefirst and second molecules can be selected include, but are not limitedto, the following binding pairs antigen/antibody, hapten/antibody,hormone/hormone receptor, sugar/lectin, biotin/avidin-(streptavidin),protein A/immunoglobulin, enzyme/enzyme cofactor, enzyme/enzymeinhibitor, enzyme/substrate, protein/modifier, and nucleic acid pairs(DNA-DNA, DNA-RNA or RNA-DNA). One example of the application ofFLASHPOINT methods is shown in FIG. 8.

[0153] Molecular Beacons

[0154] The molecular beacons used in the methods of the invention aretypically an oligonucleotide to which is attached a first label and asecond label. Most typically, the first and second label interact whenin proximity (e.g., due to resonance transfer), and the relativeproximity of the first and second labels is determined by measuring achange in the intrinsic fluorescence of the first or second label.Commonly, the emission of the first label is quenched by proximity ofthe second label. After incubation, the presence or absence of adetectable label emission is detected. The detected emission can be anyof: an emission by the first label, an emission by the second label, andan emission resulting from a combination of the first and second label.Typically, a change in the signal due to endonuclease-induced cleavageof the nucleic acid between the labels is detected (e.g., a reduction inquenching which leads to an increase in signal from either or both ofthe labels, a change in signal color, and the like). For discussion ofmolecular beacons, see, e.g., Tyagi and Kramer (1996) Nature Biotechnol.14: 303-308.

[0155] Many appropriate interactive labels are known. For example,fluorescent labels, dyes, enzymatic labels, and antibody labels are allappropriate. Examples of preferred interactive fluorescent label pairsinclude terbium chelate and TRITC (tetramethylrhodamine isothiocyanate),europium cryptate and allophycocyanin and many others known to one ofskill. Similarly, two colorimetric labels can result in combinationswhich yield a third color, e.g., a blue emission in proximity to ayellow emission provides an observed green emission. Through use ofappropriate fluorophores, molecular beacons can be made in manydifferent colors.

[0156] With regard to preferred fluorescent pairs, there are a number offluorophores which are known to quench one another. Fluorescencequenching is a bimolecular process that reduces the fluorescence quantumyield, typically without changing the fluorescence emission spectrum.Quenching can result from transient excited state interactions,(collisional quenching) or, e.g., from the formation of nonfluorescentground state species. Self quenching is the quenching of one fluorophoreby another; it tends to occur when high concentrations, labelingdensities, or proximity of labels occurs. Fluorescent resonance energytransfer (FRET) is a distance dependent excited state interaction inwhich emission of one fluorophore is coupled to the excitation ofanother which is in proximity (close enough for an observable change inemissions to occur). Some excited fluorophores interact to formexcimers, which are excited state dimers that exhibit altered emissionspectra (e.g., phospholipid analogs with pyrene sn-2 acyl chains); see,Haugland (1996) Handbook of Fluorescent Probes and Research Chemicals,Published by Molecular Probes, Inc., Eugene, Oreg., e.g., at chapter13). DABCYL, a non-fluorescent chromophore, can serve as a universalquencher for many fluorophores used in molecular beacons (Glen Research,Sterling Va.). Methods for attaching DABCYL to oligonucleotides aredescribed in Yoo et al. (1995) J. Org. Chem. 60: 3358-3364 and McMinn etal. (1996) Tetrahedron 52: 3827-3840; see also, Glen Research 1997Catalog.

[0157] The Forster radius (R₀) is the distance between fluorescent pairsat which energy transfer is 50% efficient (i.e., at which 50% of exciteddonors are deactivated by FRET. The magnitude of R₀ is dependent on thespectral properties of donor and acceptor dyes:

R₀=[(8.8×10²³)(K ²)(n ⁻⁴)(QY _(D))(J)(λ)]^(⅙) Å

[0158] where:

[0159] K²=dipole orientation range factor (range 0 to 4, K²=⅔ forrandomly oriented donors and acceptors);

[0160] QY_(D)=fluorescence quantum yield of the donor in the absence ofthe acceptor;

[0161] n=refractive index; and,

[0162] J(λ)=spectral overlap integral=∫ε_(A)(λ)·F_(D)λ·λ⁴dλcm³M⁻¹,

[0163] Where ε_(A)=extinction coefficient of acceptor andF_(D)=Fluorescence emission intensity of donor as a fraction of totalintegrated intensity.

[0164] Some typical R₀ are listed for typical donor-acceptor pairs:Donor Acceptor R₀(Å) Fluorescein tetramethylrhodamine 55 IAEDANSfluorescein 46 Fluorescein Fluorescein 44 BODIPY BODIPY 57 EDANS DABCYL33

[0165] An extensive compilation of R₀ values are found in theliterature; see, Haugland (1996) Handbook of Fluorescent Probes andResearch Chemicals Published by Molecular Probes, Inc., Eugene, Oreg. atpage 46 and the references cited therein.

[0166] In most uses, the first and second labels are different, in whichcase FRET can be detected by the appearance of sensitized fluorescenceof the acceptor or by quenching of the donor fluorescence. When thefirst and second labels are the same, FRET is detected by the resultingfluorescence depolarization.

[0167] In addition to quenching between fluorophores, individualfluorophores are also quenched by nitroxide-labeled molecules such asfatty acids. Spin labels such as nitroxides are also useful in theliquid phase assays of the invention.

[0168] Detection of Intermolecular Interactions

[0169] In the methods of the invention, a molecular beacon is attachedto a first molecule of the pair of molecules between which aninteraction is to be detected. The molecular beacons used in the methodsare typically an oligonucleotide to which is attached two labels. Asdiscussed above, the first and second labels on the molecular beacon arespaced such that, upon cleavage between the two labels, the signalresulting from the labels changes (FIG. 7). This can be easilydetermined empirically for any combination of label pairs, by cleavingthe nucleic acids using progressively longer distances between labels(i.e., by increasing the number of nucleotides between the labels), andmonitoring the resulting changes in emission properties. As noted above,the literature provides R₀ for a large number of label pairs. Typically,the first and second labels will be between about 8 and about 40nucleotides apart.

[0170] The molecular beacon can be attached to a member of the bindingpair either directly or indirectly. For example, one can covalentlyattach the molecular beacon to a molecule of interest. Methods offorming a linkage between an oligonucleotide (such as that which is acomponent of the molecular beacon) and other types of molecule are knownto those of skill in the art. One suitable method involves incorporatinginto the molecular beacon (preferably in the loop portion) an amino-dTresidue. This is then conjugated using a chemical linker to a functionalgroup (e.g., an amine group) on the molecule of interest (see, e.g.,Partis et al. (1983) J. Prot. Chem. 2: 263-277). Alternatively, themolecular beacon can be attached to the molecule of interest indirectlyby noncovalent means. For example, the molecular beacon can be attachedto a binding moiety (e.g., an antibody) that binds to the binding pairmember of interest.

[0171] According to the methods of the invention, proximity between afirst molecule and a second molecule is detected by cleavage of themolecular beacon attached to the first molecule by an endonucleasemoiety attached to the second molecule. Suitable endonuclease moieties,including those in which endonuclease activity is inducible (e.g., bycalcium) are discussed in detail above. The attachment of theendonuclease moiety to the second molecule or to the binding moiety canbe either direct (e.g., covalent) or indirect. Methods for directchemical conjugation are known to those of skill in the art and include,for example, use of a heterobifunctional linker. Suitable linkers areknown to those of skill in the art. One example of a suitable linker isthe heterobifunctional linker SMCC (succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate; Sigma Chemical Co., St. Louis, Mo.), whichcan form a link between an amino residue (for example, lysine) and athiol (such as that provided by cysteine). Other cross-linkers include,for example, m-maleimidobenzyl-N-hydroxysuccinimide ester (MBS) (Liu etal. (1979) Biochemistry 18: 690; Green et al. (1982) Cell 28: 477),glutaraldehyde, a carbodiimide succinyl anhydride,N-succinimidyl-3-[2-pyridyldithio]-propionate, and the like.

[0172] The endonuclease moiety also can be attached to the secondmolecule indirectly, e.g., by attachment to a binding moiety that canbind to the second molecule. For example, one can attach theendonuclease moiety to an antibody that binds to the molecule ofinterest. Attachment of the endonuclease moiety to either the secondmolecule of the binding pair, or to a binding moiety, should notinterfere with the enzymatic activity of the endonuclease and shouldprovide rotational freedom for the endonuclease domain. Thus, in apreferred embodiment, the endonuclease moiety is attached by way of aflexible tail such as, for example, a flexible linker. Linkers can be,for example, flexible amino acid chains that are attached to theendonuclease domain, and/or chemical linkers of suitable length. Thelinker can be of any desired length, with a shorter length resulting inhigher resolution due to the requirement for closer proximity of themolecular beacon associated with the first molecule and the endonucleaseassociated with the second molecule. In preferred embodiments, themethods can detect two molecules that are separated by approximately 50nm or less, more preferably about 10 nm or less, and most preferablyabout 1 nm or less.

[0173] Where the endonuclease moiety is to be attached to a polypeptide,a preferred method of attachment is to construct a gene that encodes afusion protein which includes both the endonuclease moiety and thebinding moiety or the molecule of interest. Methods of constructing andexpressing genes encoding fusion proteins are known to those of skill inthe art. In a preferred embodiment, the chimeric gene also encodes aflexible polypeptide linker between the endonuclease moiety and thebinding moiety.

[0174] The FLASHPOINT methods of the invention are useful for detectingintermolecular interactions in vitro, in situ, and in vivo. For example,one can use the methods to detect intermolecular interactions that occurwithin a cell, tissue, or organism. In some embodiments, the cells ortissues can be fixed, embedded, and/or sectioned by methods known tothose of skill in the art (see, e.g., Ausubel et al., supra.). Themolecular beacon and the endonuclease domain are added to the sample andallowed to bind to their respective target molecules. In preferredembodiments in which an inducible endonuclease moiety is used,endonuclease activity is induced after binding has occurred. If thetarget molecules are in close proximity, the endonuclease cleaves themolecular beacon, thus resulting in emission of a signal. The signal canbe localized using, for example, fluorescence microscopy.

[0175] The invention also provides methods by which one can determinewhether the absence of a signal is due to poor binding of the chimericendonuclease and/or the molecular beacon to their respective targetmolecules, rather than being due to the target molecules not being inclose proximity. In these embodiments, either or both of the chimericendonuclease and the molecular beacon-containing moiety are labeled witha fluorophore of different excitation and emission wavelengths than thebeacon. These labels are then visualized, e.g., with a fluorescencemicroscope. The presence of a signal from the fluorescent labelsattached to the chimeric endonuclease and the molecularbeacon-containing moiety, but not a signal from the molecular beaconitself, indicates that the target molecules are not in close proximity.

[0176] Amplification of Signals from Immunohistological Methods

[0177] Also provided by the invention are methods of obtaining greatersensitivity in immunohistological techniques. A schematic diagram ofthese methods is shown in FIG. 9. The methods offer significantadvantages over previously known methods for detecting target molecules,particularly where localization of target molecules is desired. Highresolution localization of a molecule by microscopy, for example, istypically performed using confocal laser microscopy. This requires theuse of fluorophore-tagged antibodies; because the fluorophores aredirectly conjugated to the antibodies, the emitted fluorescence cannotbe amplified, thus resulting in a relatively weak signal in many cases.The methods of the invention overcome this drawback by providing a meansby which a fluorescent signal can be amplified, thus increasingsensitivity.

[0178] In the methods of the invention, a target molecule is contactedwith a chimeric endonuclease which binds to the target molecule. Thechimeric endonuclease is then contacted with a molecular beacon. Theendonuclease can cleave multiple molecular beacons that come intocontact with the endonuclease moiety, thus resulting in amplification ofthe fluorescence signal. To maximize the signal, fluorescence isintegrated over time.

[0179] In a typical embodiment, the methods involve preparation of aslide of tissue or cells and treating the slide with a primary antibody,as is done in conventional immunofluorescence assays. Instead ofdetecting the primary antibody by use of a fluorophore-tagged antibody,for example, the primary antibody is contacted with a detection moiety,termed an “immunopointer,” that binds to the primary antibody andincludes an endonuclease domain, preferably one that is inducible. Uponbinding of the immunopointer to the primary antibody, a molecular beaconis added and endonuclease activity is induced.

EXAMPLES

[0180] The following examples are provided by way of illustration onlyand not by way of limitation. Those of skill will readily recognize avariety of noncritical parameters which could be changed or modified toyield essentially similar results.

Example 1

[0181] Identification of Protein Position on Target Plasmid DNA (FIG. 1)Using a Chimera Comprising a Micrococcal Endonuclease Domain(Pinpoint-MNase).

[0182] In one embodiment, the invention provides methods of cleaving anddetecting a target plasmid nucleic acid in vivo. This is useful, interalia, for monitoring the interaction between a chimeric guide proteinand a selected target nucleic acid. For example, the interaction betweena transcription factor guide domain and a promoter target nucleic acidcan be monitored. This facilitates the study of gene expression, andprovides a basis for screening for the effect of potential therapeuticagents on transcription factor activity. For example, using ap53-endonuclease chimera, it is possible to screen for agents whichmodulate p53 activity.

[0183] An expression vector encoding a chimeric guide-micrococcalendonuclease fusion protein is transduced or injected into cells. Thechimeric fusion protein optionally comprises a oligonucleotide regionbetween the guide and micrococcal domains, such as a polyglycinesequence.

[0184] Approximately 16-48 hours after transduction, the cells areisolated and transduced for a second time with the target plasmid DNAcontaining a potential target site recognized by the chimericguide-micrococcal endonuclease fusion protein. Approximately 2 to 48hours after the second transduction the cells are permeabilized with amild detergent such as lysolecithin or NP-40 in the presence of 2 mMexogenous calcium for 1-10 minutes to activate the MNase domain on theexpressed chimeric protein. The target DNA (along with other lowmolecular weight DNA) is recovered using a standard Hirt preparationprotocol. See, e.g., Anant and Subramanian (1992) Methods in Enzymology216, 20-29.

[0185] The nick created by the chimeric protein (called the “targetcleavage site” or “point”) in the target nucleic acid is converted to adouble stranded blunt end by primer extending with an oligonucleotideprimer (see, FIG. 1). The blunt end is marked by ligating a trappingoligonucleotide after phosphorylating the 5′ OH end created by the MNasedomain of the chimeric protein. A fragment marked by ligation of thetrapping oligonucleotide is amplified with PCR using an internal primerand a second primer which is derived from a sequence in the trappingoligonucleotide. The amplified fragment is then detected with one of anumber of different strategies, such as primer extension with alabelling primer (e.g., radioactively or fluorescently labelled),Southern blot analysis, or nested PCR. In many cases, primer extensionwith the labelled oligonucleotide primer is sufficient to detect thepoint.

[0186] One way of marking the 3′ end of a point is to extend it withterminal transferase after dephosphorylating it. A dephosphorylationstep is used because MNase leaves a 3′ phosphate end, which cannot beextended by terminal transferase or by ligation. The 3′ end can also bemarked by ligating a single stranded oligonucleotide. Fragments thusmarked are PCR amplified with a primer made complementary to thesequence added on the 3′ end, and a primer from a known internal site.

Example 2

[0187] Identification of a Position on a Known Genomic Region Using aChimera Comprising a Micrococcal Endonuclease Domain.

[0188] In one embodiment, the invention provides methods of cleaving anddetecting a target nucleic acid from a known genomic sequence in vivo.This is useful inter alia, for determining whether a selected genomicnucleic acid is bound by a particular chimeric guide protein. Forexample, the interaction between a transcription factor guide domain anda promoter target nucleic acid can be monitored. This facilitates thestudy of gene expression, and provides a basis for screening for theeffect of potential therapeutic agents on transcription factor activity.For example, using an Sp1-endonuclease chimera, it is possible to screenfor agents which modulate Sp1 activity. Similarly, the effects of anypotential modulator of a transcription factor oncogene such as c-myc canbe assessed.

[0189] The expression vector for the chimeric guide endonuclease fusionprotein is either transiently or stably transduced into cells (see, FIG.2). If cells are transiently transduced, the chimeric guide endonucleasefusion protein expression vector is cotransduced with an expressionvector for a marker gene such as GFP (green fluorescent protein). Cellsthat express GFP, and therefore most likely express the chimeric guideendonuclease fusion protein, are isolated by FACS (fluorescenceactivated cell sorting) or HOOK.

[0190] This step facilitates selection of transiently transduced cells.Transduction efficiency for most cells, and most transduction proceduresis approximately 10% and, therefore, a selection step as described isused to ensure that a higher percentage of cells under analysis includethe chimeric guide endonuclease fusion protein. Alternatively, aselection step is not required for stable cell lines since every cellexpresses the chimeric guide endonuclease fusion protein.

[0191] After chimeric guide endonuclease fusion protein expressing cellsare isolated, they are permeabilized with a mild detergent such aslysolecithin or NP-40 and incubated for 2 to 5 minutes in calciumactivation buffer to activate the MNase before adding a stop buffer(containing EDTA or Zinc). Subsequent steps starting with primerextension are similar to Example 1, with the additional step of physicalisolation of DNA fragments prior to PCR. Although different strategiesare possible, labelling the trapping oligonucleotide with biotin andisolating trapped DNA fragments with streptavidin magnetic beads is afavored way of isolating DNA fragments. This isolation step decreasesthe background signal from PCR amplification of unmarked DNA.

Example 3

[0192] Identification of Chimeric Guide Endonuclease Fusion ProteinBinding to an Unknown Genomic Target (Differential PINPRINT)

[0193] PINPOINT-MNase can be used to scan all genomic positions of achimeric guide endonuclease fusion protein simultaneously. The chimericguide endonuclease fusion protein is expressed and expressing cellsisolated as described in Example 2. In order to minimize shearing whichcan produce unwanted background of cleaved sites, the genomic DNA isisolated while the cells are embedded in an agarose plug. Afterpurification, agarose-embedded genomic DNA is digested with arestriction enzyme such as Nco 1 to minimize shearing in subsequentsteps. Since MNase created cleavage sites are distinguished fromendogenous nuclease induced cleavage sites by the 3′ P and 5′ OH endsthat MNase generates, it is important to reduce shearing as much aspossible, because shearing of the DNA can also create 3′ P and 5′ OHtermini.

[0194] Although the digestion with Nco1 creates cleaved ends, these areeliminated later based on the Nco 1 recognition sequence, and,therefore, are not problematic. Since the nicks created by MNase cannotbe ligated using a standard ligase, a ligase is added to seal nicks thathave 5′ P and 3′ OH ends to reduce background in subsequentamplification reactions.

[0195] Any nicks in the genomic DNA created by the chimeric guideprotein are converted to double stranded blunt ends with primerextension using random primers (see, FIG. 3). The blunt ends having 5′ Pand 3′ OH ends are blocked by ligating blocking linker oligonucleotides,which only ligate to ends with a 5′ P, leaving MNase specific nicksintact. At this point, only the ends with the 5′ OH are available forligation. After phosphorylation of the 5′ OH end, a biotinylatedtrapping linker oligonucleotide is ligated to the resulting 5′ P.Unligated trapping oligonucleotide is discarded. After the trappedfragments are isolated as above (see, Example 2) using streptavidinbeads, they are cleaved with a pair of 4 base cutting restrictionenzymes (such as Alu1 and Rsa1) that leave blunt ends. A reaching linkeroligonucleotide is then ligated to the blunt ends. The fragments thatare bracketed with a trapping oligonucleotide and a reachingoligonucleotide are amplified by PCR using a primer that iscomplementary to the trapping linker oligonucleotide and a primer thatis complementary to the reaching linker oligonucleotide.

[0196] Since the positions of the 4 base cutting restriction enzymes aredifferent relative to each nick created by the chimericguide-endonuclease fusion protein, electrophoresis of the amplifiedfragments results in a ladder pattern on the electrophoretic gel. Bycomparing ladders for different chimeric guide-endonuclease fusionproteins side by side, one can distinguish chimeric guide-endonucleasefusion proteins specific bands from non-specific bands. The specificbands are cut out of the gel, reamplified with PCR and cloned.

[0197] A similar strategy for identifying the 3′ side of a targetcleavage site using either terminal transferase extension or oligoligation is shown on the right side of FIG. 3. In this protocol, the 3′OH generated by the restriction enzyme (e.g., Nco1) is extended usingeither a terminal transferase, or a blocking oligonucleotide. After 3′dephosphorylation of the 3′ phosphate at the target cleavage site madeby the chimeric guide-endonuclease fusion protein, the 3′ end isextended with a terminal transferase, or by ligating an extensionoligonucleotide to the end. Double-stranded DNA is made by primerextension using a biotynylated oligonucleotide complementary to theblocking oligonucleotide, or complementary to the end made usingterminal transferase. A restriction enzyme with a four base recognitionsite is used to cleave the double-stranded DNA. The resulting cleavedDNA is isolated on streptavidin beads. A reaching oligonucleotide linkeris ligated to the isolated DNA, and the isolated DNA is amplified usingprimers complementary to the reaching linker and complementary to thebiotinylated oligonucleotide. This amplified DNA is detected and/orcloned for subsequent analysis.

Example 4

[0198] Identification of Protein Position on Unknown Genomic Target(Point Directory)

[0199] A more comprehensive way to determine the genome-wide positionswhich are cleaved by a chimeric guide-endonuclease fusion protein is tocreate a “point directory,” i.e., a library of nucleic acidscorresponding to many of the target cleavage sites (points). The stepsfor this technique is same as that of Example 3 until ligation of thetrapping linker oligonucleotide (see, FIG. 4). After phosphorylation ofprimer extended DNA, a blocking linker oligonucleotide is ligated toblock all ends having a 5′ P. Half of the sample is ligated to trappinglinker oligonucleotide A and the other half is ligated to trappinglinker oligonucleotide B. These two trapping oligonucleotides containthe recognition sequence for a class IIS restriction enzyme, such asBsg1, which cleaves 16 bp 3′ to the enzyme's recognition sequence. Thus,cleaving the trapped DNA with Bsg1 leaves a 16 bp subsequence (termed an“address”) of the target cleavage site attached to the respectivetrapping linker. Fragments cleaved by the class IIS restriction enzymeare physically isolated, e.g., with magnetic streptavidin beads.Isolated DNA fragments are then separated from the magnetic beads bycleaving within the trapping linker with a restriction enzyme whosecleaves site is found within the linker (e.g., Nru1). The liberated DNAfragments attached to the two trapping linkers are then ligatedtogether. The ligated fragments are amplified with PCR using primers Aand B. The amplified fragments are cleaved with a second restrictionsite found within the cleaved linker (e.g., Not 1) and ligated into aconcatemer and cloned into a plasmid vector such as the universallyavailable pBluescript II. Plasmid DNA containing the concatemers isisolated and sequenced from the opposite ends of the concatemer. Theconcatemers will contain two point-addresses separated by a restrictionsite (e.g., a Not 1 site).

[0200] The addresses are compiled and the location identified bysearching the genomic sequence databases, or by using the addresses tomake probes to screen genomic libraries.

[0201] PINPOINT is useful not only for identifying positions of staticproteins bound to DNA or near DNA (i.e. tethered by protein-proteininteraction), but those with processivity as well. Because the positionof a processive RNA polymerase is reflective of the transcriptionalstatus of that gene, one can compile the point-addresses of RNApolymerase, and therefore its transcriptional status, using thetechniques above. The conventional analysis of the steady statecytoplasmic RNA levels, e.g., by northern analysis, reflects not onlytranscription but posttranscriptional modification (such as splicing),RNA transport and stability, and does not reliably reflect thetranscriptional status of a promoter at any given time. In order tounderstand gene expression, it is useful to have the capability tomonitor all variables at play at any given moment during geneexpression, starting from the recruitment of transcription factors. Thisability is provided by the above techniques.

[0202] MNase also cleaves RNA in the presence of calcium, leaving a 5′OH and 3′ P ends. After reverse transcription, strategies similar tothose described for DNA are applied to detecting the positions of RNAbinding proteins such as one of EIF-4 proteins which are important fortranslation of mRNA. By compiling a directory of the EIF-4 addresses,one can determine the translational status of all the mRNA in a cell atany given moment.

Example 5

[0203] PINPOINT-Fok

[0204] Like MNase, the nuclease domain of Fok1 creates single strandednicks on DNA in vivo; unlike MNase, however, it leaves a 5′ P and 3′ OHat the cleavage site. These ends are extended with terminal transferasewithout dephosphorylation of the 3′ end, or ligated directly to atrapping linker oligonucleotide without phosphorylation of the 5′ end,after primer extension and Klenow treatment, or Klenow treatment alone.Identification of the points created by the nuclease domain of Fok 1 canbe done as described for MNase above.

Example 6

[0205] PINPOINT for RNA Point Detection (FIG. 5)

[0206] Since MNase also cleaves RNA in the presence of calcium, leavinga 5′ OH and a 3′ P end, PINPOINT strategies are also used to identifyRNA binding sites for both known and unknown RNA binding proteins. Thestrategies are similar to those described for DNA binding proteins inFIGS. 1 through 4 and examples 1-5, with a few modifications. Thestrategies that correspond to the DNA strategies in FIGS. 1 and 2 aredifferent primarily in that they use a primer of known sequence forreverse transcription.

[0207] After RNA cleavage by a chimeric protein comprising an MNasecleavage domain which cleaves RNA is induced with calcium, RNA isisolated and all the 3′ OH ends are first blocked with T4 RNA ligase andterminating ribose nucleotides such as 3′-O-Methylguanosine 5′Triphosphate. Once the terminating nucleotide is added to the 3′ end, itis no longer available for further ligation or extension. The remaining3′ P ends, created by MNase, are dephosphorylated with T4 polynucleotidekinase or a phosphatase enzyme. The 3′ OH ends are marked by ligatingprimer A with RNA ligase or half of the sample with primer A and halfwith primer B for differential PINPRINT or point directory strategiesoutlined in the examples above, respectively. Using a biotinylatedprimer complementary to the ligated primer, double stranded cDNA ismade. The biotinylated DNA fragments are isolated with streptavidinbeads as described earlier. The remaining steps for both differentialPINPRINT and point directory are identical as for identifying points onDNA.

Example 7

[0208] ImmunoPINPOINT

[0209] In one aspect, the present invention provides chimeric proteincomprising an antibody, or a recognition domain derived from anantibody, attached via a linker domain to a micrococcal endonucleasedomain. The antibody recognizes either a protein which binds, directlyor indirectly, to a nucleic acid, or a primary antibody which binds,directly or indirectly, to the nucleic acid.

[0210] Cells comprising a target nucleic acid are fixed andpermeabilized. The fixed, permeabilized cells are then incubated with achimeric guide endonuclease protein, where the guide domain is derivedfrom an immunoglobulin. In embodiments where the immunoglobulinrecognizes a protein bound to a nucleic acid, the immunoglobulin guidedomain binds to the protein bound to the nucleic acid, and positions thechimeric protein near the binding site. After calcium activation, thecleavage domain cleaves the nucleic acid, which is then amplified ordetected in the same manner as described supra. See also, FIG. 6.

[0211] In embodiments where the immunoglobulin guide domain recognizes aprimary antibody, the cells are incubated with both the chimeric proteinand the primary antibody. The primary antibody binds to a protein boundto the nucleic acid and the immunoglobulin guide domain positions thechimeric protein near the binding site by binding to the primaryantibody. After calcium activation, the cleavage domain cleaves thenucleic acid, which is then amplified or detected in the same manner asdescribed supra. See also, FIG. 6.

[0212] Although this example is described for an application using animmunoglobulin guide domain, it will be appreciated that similar in situmethods are also provided for guide domains other than antibodies bysubstitution of alternate guide domains in the method.

Example 8

[0213] PINPOINT Analysis of the Globin LCR

[0214] A locus control region (LCR) confers high level,position-independent expression of a linked gene (see, Orkin (1995) EurJ Biochem 231:271-81). The best characterized among these is theβ-globin LCR which serves as the master regulatory element for theexpression of the globin family of genes in a locus that spans almost100 kb (Orkin, id.). The expression of the globin family of genes inerythroid cells are developmentally regulated. In human, the ε-globingene is expressed first in the embryo, followed by the gamma globingenes in the fetus, and the β-globin gene at birth and throughout life.Proper expression of all these genes is dependent on the LCR, whichresides in four DNase 1 hypersensitive sites (5′HS1-5) located upstreamof the ε-globin gene. The complete set of these hypersensitive sites isrequired for full position-independent expression suggesting that theyact synergistically. These DNase1 hypersensitive sites contain a numberof binding sites for CACCC and E box binding factors, GATA-1 and NFE-2as well as yet uncharacterized factors (Talbot and Grosveld (1991) EMBOJ. 10:1391-8).

[0215] The LCR-globin promoter interaction, presumably mediated by thesefactors for which binding sites are present in the LCR, appears to bedynamic during development. For example, LCR-mediated expression shiftsback and forth between two genes (e.g., gamma- and β-globin) during theswitch from gamma-globin to β-globin gene expression before settling onstable β-globin expression. The LCR-linked β-globin gene is expressedeven when it is embedded in heterochromatin, suggesting that the LCRalso opens the chromatin, perhaps by recruiting chromatin modifyingcomplexes such as NURF or BRG1. Indeed, in Hispanic thalassemia wherethe LCR is deleted, the chromatin of the whole β-globin domain iscondensed. As a result, the promoter regions, which are nucleosome freein the presence of the LCR, are occupied by nucleosomes. Perhaps as arelated phenomenon, the β-globin domain no longer replicates early in Sphase but late.

[0216] The molecular mechanism by which the β-globin LCR activatestranscription is not known, but a number of studies have shown thatminimal promoter elements play an important role (Antoniou and Grosveld(1990). Genes Dev 4:1007-13). The β-globin promoter region can bedivided into two regions, upstream (−110 to −815) and minimal (1 to−110). The upstream region contains, among others, GATA-1 and NF 1binding sites and a CAAT box. The minimal promoter region contains twoCACCC (distal and proximal), one CAAT and one TATA boxes. In thepresence of the LCR, the minimal promoter is sufficient for maximalexpression. Mutation of the proximal CACCC box and or CAAT box severelyweakens the LCR induced β-globin promoter activity. The role of theproximal CACCC box is further demonstrated by the studies in thalassemiapatients which show clustering of point mutations in the proximal CACCCbox and the TATA box, resulting in severely decreased β-globinexpression. A point mutation in the distal CACCC box resulted in only aslight decrease in β-globin expression Weatherall et aL (1989) “TheHemoglobinopathies” In The Metabolic Basis of Inherited Disease, ed.Scriver, et al. 2281-2339. New York: McGraw-Hill, 6th ed).

[0217] These results raise several questions about the signals thatcontrol globin gene regulation. For example, the presence of multiplecopies of the CACCC box in the LCR itself raises the question of why anadditional CACCC box in the minimal promoter is required. Similarly,because the promoter and its upstream region contain binding sites fortranscription activators, it is unclear why the LCR is necessary. Invitro experiments on naked DNA have shown that transcription activatorssuch as CACCC box binding factors recruit and stabilize preinitiationcomplex (PIC) and or RNA polymerase II holoenzyme throughprotein-protein interaction (Pugh and Tjian (1990) Cell 61:1187-97;Chiang and Roeder (1995) Science 267:531-6; Gill et al. (1994) ProcNatl. Acad. Sci. U S A 91:192-6; Stargell and Struhl (1996) TrendsGenet. 12:311-5; Struhl (1996) Cell 84:179-82). In chromatin, however,such transcription activators must compete with the histone octamers forthe binding site. The success in such competition will depend on thelocal chromatin structure. Furthermore, as has been demonstrated withother transcription activators (Elefanty et al. (1996) EMBO J. 15:319-33), the local concentration of CACCC box binding factors in vivo islikely to vary from one region of the nucleus to another and, as aresult, the likelihood that a CACCC box is occupied will also likelydepend on its nuclear position. Therefore, in order to confer highlevel, position independent expression, the LCR must efficiently recruittranscription activators such as the CACCC box binding factor regardlessof the local concentration and chromatin structure. Since thetranscription factors and histones compete for DNA, the resulting highoccupancy rate of the CACCC box should also prevent reformationnucleosome at the promoter region.

[0218] In order to understand the mechanism by which the LCR mediatesthe ε- to γ- to β-globin switches and position-independent expression ofthe β-globin genes, it is necessary to first understand, at themolecular level, how the LCR influences, if at all, the recruitment ofthe various polypeptides involved in β-globin gene expression (i.e.transcription activators and general transcription factors). However,for reasons discussed above, in vitro methods for analyzingprotein-nucleic acid interactions do not accurately reflect theconditions that actually exist in vivo. To circumvent these shortcomingsof previously available techniques, the strategy described herein whichemploys chimeric guide-endonucleases to analyze these interactions.

[0219] This Example describes the use of PINPOINT techniques to test thehypothesis that the globin LCR promotes efficient recruitment of theCACCC box binding factor to the human β-globin promoter. At least fourpolypeptides bind the CACCC box in erythroid cells; three of them, Sp1,BKLF and EKLF have been cloned (Kadonaga et al. (1987) Cell 51:1079-90;Crossley et al. (1996) Mol Cell Biol. 16:1695-705; Miller and Bieker(1993) Mol Cell Biol. 13: 2776-86). Proper expression of the β-globingene requires EKLF, but it is not known whether this is a direct orindirect effect (Nuez et al. (1995) Nature 375:316-8; Perkins, et al.(1995) Nature 375:318-22). In cotransfection experiments, all three ofthese polypeptides can activate expression of the β-globin gene. SinceSp1 is the best characterized among these three factors, we have chosento focus on in vivo recruitment of Sp1.

[0220] A. Material and Methods

[0221] 1. Construction of Plasmids.

[0222] A chimeric endonuclease that includes a FokI cleavage domainlinked to a DNA binding domain from the transcription factor Sp1 wasobtained as follows. A pCMV expression vector containing an Sp1-FokIfusion gene under the control of a CMV enhancer/promoter was constructedas follows. The cleavage domain of FokI restriction endonuclease (Li andChandrasegaran (1993) Proc Natl Acad Sci U S A 90:2764-8) was placeddownstream of the pCMV enhancer/promoter. DNA encoding a nine glycineresidues linker was inserted at the 5′ end of the FokI fragment.

[0223] DNA fragments encoding various fragments of Sp1 were insertedinto this vector upstream of the glycine linker and the cleavage domainof Fok I, under the operable control of the CMV promoter. A DNA fragmentencoding Sp1 amino acids 83-685 (which includes the DNA binding domain)was inserted at the 5′ end of the glycine linker. The vector pCMV-ABCSp1-FokI was constructed by inserting DNA encoding Sp1 amino acids83thr-530asp at the 5′ end of the glycine linker. The D domain of Sp1(620 gln through C-terminal) was inserted between ABC of Sp1 and FokI toobtain the pCMV-ABCD Sp1-Fok I expression vector. PCMV-AB Sp1-Fok I wascreated by deleting the region encoding the Sp1 C domain from pCMV-ABCSp1-Fok I, in order to express the N-terminal to the 363arg amino acidresidue of Sp1. In another construct, a Gal4 binding domain was placedupstream of the glycine-encoding linker and the cleavage domain of FokI,to create Gal4-FokI.

[0224] For use as reporter genes, the human β-globin promoter (−374 to+21) was linked to a gene encoding chloramphenicol acetyltransferase(CAT). Various portions of the globin LCR were placed upstream of theβ-globin promoter as follows. pHS234 (p269) was made by insertingupstream of the β-globin promoter a 4.63 kb HpaI to SalI fragment frompHS 1234 (derived from LAR-β, see, Forrester et al. (1989) Proc NatlAcad Sci U S A 86:5439-43) that contains HS 2, 3, and 4 of the globinLCR. Additional LCR combination target DNAs were derived from pPN86(Amrolia et al. (1995) J Biol Chem 270:12892-8): pHS23 was constructedby inserting a 0.85 kb SacI fragment from pHS1234 that included HS 3into the SacI site of pPN86(HS 2) (Amrolia, Id.), pHS2,4 was constructedby inserting a 1.25 kb SacI fragment of HS4 from pHS1234 which wasblunt-ended by Klenow fragment, into the XhoI site of pPN86 that alsohad been blunt-ended using Klenow fragment; pHS34 was constructed byinserting a 2.1 kb BamHI to SalI fragment of pHS 1234 that contains HS3and 4 into BglII-XhoI cleaved pPN86.

[0225] Promoter-deletion mutant target DNAs were constructed as follows.The promoter region was first sectioned as a GATA-1 binding site (−374to −112), a CACCC binding site (−111 to −77), a CAAT box (−76 to −30),and a TATA box (−30 to +21). pHS234 was used as the wild type (GCCT),which contains each of these sections. PCR primers specific for eachsection were used to make the promoter deletion mutants. The “CCT”mutant, which contains a CACCC binding site, a CATTT box, and a TATAbox, but lacks a GATA-1 binding site, was generated by inserting DNAfragments amplified from the this region of pHS234 using primer 2(linked to an 18 mer containing a NotI site;5′-AAGGAAAAAAGCGGCCGC-Region 2-3′ (SEQ ID NO:1)) and JS52(5′-CGTGGTATTCACTCCAGAGCGATGAAAAC-3′ (SEQ ID NO:2)). The amplifiedfragment was digested with NotI and EcoRI and inserted into NotI- andEcoRI-digested pHS234.

[0226] The “CT” deletion mutant, which contained a CAT box and a TATAbox, was made by inserting a DNA amplified using primer 3 (Region 3linked to an 18 mer NotI linker;(5′-AAGGAAAAAAGCGGCCGCGGCCAATCTACTCCCAGGAGCAG-3′; (SEQ ID NO:3)) andJS52 into the NotI and EcoRI sites of pHS234.

[0227] The “T” mutant, which contained only the TATA box, was created byinserting an amplified DNA obtained using primer 4 (Region 4 linked toan 18 mer NotI linker;(5′-AAGGAAAAAAGCGGCCGCATAAAAGTCAGGGCAGAGCCATCTAT-3′; (SEQ ID NO:4)) andJS52; the fragment was digested with NotI and EcoRI and inserted intoNotI- and EcoRI-digested pHS234.

[0228] The “GCC” mutant, which had a GATA-1 binding site, a CACC bindingsite, and a CAT box, but lacked a TATA box, was generated by inserting aDNA amplified using primer 1(5′-AAGGAAAAAAGCGGCCGCAGCTCTTCCACTTTTAGTGCAT-3′; (SEQ ID NO:5)) andprimer 5 (Region 5 linked to nonamer containing EcoRI site;5′-CCGGAATTCGCCCAGCCCTGGC-3′ (SEQ ID NO:6) that had been treated withNotI and EcoRI into NotI- and EcoRi-digested pHS234.

[0229] The “CC” mutant, which had a CACC binding site and a CAT box, wasconstructed by inserting amplified DNA produced with Primers 2 and 5that had been treated with NotI and EcoRI into NotI- and EcoRI-digestedpHS234.

[0230] 2. Transfections

[0231] Transient cotransfection of murine erythroleukemia (MEL) cellswas performed with 1-10 μg of expression vector and 3-5 μg target DNA.Electroporation was performed at 975 μF, 250 V, and resistance level 5(BTX Electro Manipulator 600) with about 10⁷ cells in 0.7 ml of Dulbeccomodified Eagle medium without serum. The cells were immediately platedin 20 ml of DMEM containing 10% serum.

[0232] 3. DNA Isolation and Analysis.

[0233] DNA was isolated from transfected cells using the Hirt lysisprocedure (See, e.g., Anant and Subramanian (1992) Methods in Enzymology216: 20-29) 16-48 hour following transfection. Ligation-mediated PCR wasperformed as follows (see, e.g., Mueller and Wold (1989) Science 246:780-686 for general protocol). DNA isolated from the cells was treatedwith the large fragment of DNA polymerase (New England Biolabs, BeverlyMass.) in a reaction mixture containing 33 mM each dNTP in order togenerate blunt ends at the cleaved sites. Following phenol-chloroformextraction and filtration through Microcon-50™ (Amicon), blunt-ended DNAwas purified. Linker LF1/JS21B (5′-GAAACACTTCAGATCTCCCGAGTCACCGC-3′ (SEQID NO:7) annealed to 5′-phosphorylated GCGGTGACTCGGGAGATCTGAAGTG-3′ (SEQID NO:8); 50 pmol) was ligated to blunt-ended sites using 4 U of T4 DNAligase (NEB) at 16° C.

[0234] Ligation products were amplified by 25 cycles of PCR usinglinker-specific primer JS21B (5′-GAAACACTTCAGATCTCCCGAGTCACCGC-3′ (SEQID NO:9)) plus a target-specific primer JS52(5′-CGTGGTATTCACTCCAGAGCGATGAAAAC-3′ (SEQ ID NO:10)) within the CATgene. DNA was completely denatured for 3 min at 95° C. immediatelybefore PCR. Each cycle comprised 15 s at 94° C., 30 s at 69° C., and 45s at 72° C. (in a Power Block System™ of Ericomp located in San Diego,Calif.). One final cycle of extension reaction was done for 7 min at 72°C. The PCR products were run on the Nusieve™ 3:1 agarose gel (RocklandMd.) at 4.5-5.0 V/cm for 4 hour and transferred to Hybond-N+ (Amersham,Arlington Heights, Ill.). Southern hybridization was carried out at 52°C. with QuikHyb™ Hybridization Solution (Stratagene, La Jolla, Calif.).Southern blots were probed with a 17 bp CAT gene fragment (JS14;GTGAATAAAGGCCGGAT-3′ (SEQ ID NO:11)).

[0235] Primer extension analysis was performed using 5′-end-labelledJS42 (TACGATGCCATTGGGATATATCAACGGTGG-3′ (SEQ ID NO:12)) as a primer;this sequence is found at the 3′ end of the β-globin promoter. BecauseGCC and CC promoter deletion mutants do not include the region to whichJS42 would hybridize, another primer (JS52;5′-CGTGGTATTCACTCCAGAGCGATGAAAAC-3′ (SEQ ID NO:13)) was used for primerextension of all promoter deletion mutants. Template DNA was completelydenatured by heating with 10% DMSO at 94° C. for 2-3 min and quicklycooling on the dry ice. One cycle of primer extension was performed for5 min at 94° C., 5 min at 70° C., and 5 min at 72° C., with Vent™Exonuclease-Polymerase (New England Biolabs, Beverly, Mass.). Theextended products were run on a 6% TBE-Urea sequencing gel (NationalDiagnostics, Atlanta, Ga.) at 70 Watts. DNA treated with HindIII orEcoRI was used for quantitation of recovered DNA following Hirt lysis byprimer extension.

[0236] B. PINPOINT Analysis of Sp1 Recruitment to β-globin LCR

[0237] To test the hypothesis that the globin LCR recruits Sp1 to theβ-globin promoter requires the capability to visualize in vivoprotein-DNA interactions. Toward this end, the inventor developed astrategy referred to as PINPOINT-1 (Protein Position Identification withNuclease Tail-1). This strategy makes use of the chimeric endonucleasesdescribed herein. In the experiments described in this Example, anexpression vector that encodes a chimeric endonuclease composed of theSp1 transcription factor linked to the nuclease domain of type IISendonuclease Fok1 via a flexible linker, was transfected into MEL(murine erythroleukemic line) cells, along with target DNA. Fok 1 has aDNA binding domain which binds to the recognition sequence for theenzyme, and an independent nuclease domain which makes a double strandcut at a defined distance, 9 bp for one strand and 13 bp for the other,to one side of the recognition site (Li and Chandrasegaran, supra). TheSp1-FokI fusion protein, which is termed the Sp1 “pointer,” competeswith the endogenous pool of Sp1 and the other CACCC box factors inbinding to the CACCC site in the β-globin promoter. If the Sp1 pointerbinds to the CACCC box, the attached nuclease will cleave the nucleicacid near the binding site. Because the nuclease domain of FokI does nothave sequence specificity of its own, the position and the probabilityof the cleavage is determined by the position of the linked Sp1 and itslingering time at that position, respectively. The target DNA, bothcleaved and uncleaved, was harvested using the Hirt procedure and theposition of the nuclease-induced cleavage was analyzed by primerextension directly, or by ligation mediated PCR followed by primerextension with radioactively labeled internal primer.

[0238] 1. The LCR Helps to Recruit Sp1 to the β-globin Promoter.

[0239] To determine whether the β-globin LCR plays a role in recruitingSp1 to the β-globin promoter, an Sp1 pointer-expressing vector wascotransfected with one of two target plasmids: p269, which contains thehuman β-globin promoter joined to 5′ HS 2,3 and 4 of the human β-globinLCR; and p306, which contains the β-globin promoter joined to a fragmentof S phage DNA, as control. The 5′ HS 1 was not included in the LCRfragment because its deletion in a patient did not affect β-globin geneexpression. Sp1 (Kadonaga et al. (1987) Cell 51:1079-90; Kadonaga et al.(1988) Science 242:1566-70; Pascal and Tjian (1991) Genes Dev 5:1646-56)is linked to the nuclease. Low molecular weight DNA was harvested fromtransfected cells after 16 to 48 hours, and cleavage was detected usingprimer extension. The Sp1 pointer cleaves within the β-globin promotermuch more efficiently (greater than 20 fold) when the promoter is joinedto the LCR than when the promoter was joined to S phage DNA. The Sp1pointer cleavage sites are located 5 bps upstream and 10 bps downstreamof the double CACCC site in the β-globin promoter.

[0240] The simplest interpretation of this finding is that the LCRpromotes recruitment of the Sp1 pointer to the β-globin promoter, butother explanations are possible. The LCR containing target DNA, which issupercoiled when transfected, could be relaxed or linearized moreefficiently than the control target DNA. This might result if endogenousnucleases and/or Sp1 pointer is recruited efficiently to the LCR (whichcontains a number of Sp1 binding sites). If the nuclease domain of FokIis sensitive to the superhelicity of the target DNA, as someendonucleases are, it is possible that the control target DNA will notbe cleaved as well as the LCR-containing target DNA even though the Sp1pointer was recruited to the promoter of the control DNA equally as wellas to the LCR containing target DNA. To examine this possibility, thetarget DNAs were maximally relaxed with topoisomerase I beforetransfection. Again, the LCR-containing target DNA was cleaved at thepromoter much more efficiently than the control target DNA, ruling outthe possibility that DNA topology was responsible for the difference incleavage.

[0241] These results collectively support the hypothesis that the LCRrecruits Sp1 to the β-globin promoter of a transfected target DNA. Theexperiments may mimic recruitment that occurs during a brief window oftime during which the endogenous β-globin gene is being replicated andthus is free of histones. Newly replicated DNA is thought to be free ofhistones immediately following passage of the DNA replication fork, sojust after replication of the β-globin locus transcription factorrecruitment could take place.

[0242] MEL cells can be induced to differentiate and express high levelsof the β-globin gene with DMSO. DMSO induction was tested for enhancedrecruitment of Sp1 pointer to target DNAs with or without the LCR, butwas found to not enhance recruitment. This suggests that DMSO inductionaffects another step in the process leading to high levels of theβ-globin gene transcription. Such step might be part of the formation ofthe preinitiation complex (PIC) or a postinitiation process such asphosphorylation of the carboxy terminal domain (CTD) of the RNApolymerase II which is thought to stimulate elongation.

[0243] Since Sp1 easily forms a homotypic multimer, most likely atetramer, an Sp1-DNA complex may be composed of two types of Sp1: thosethat are directly bound to DNA and those that are tethered throughSp1-Sp1 interaction (see also, Pascal and Tijian, supra.). Three Sp1regions, A, B and D participate in this interaction. The cleavagepattern reflected the recruitment of both Sp1 types, since the Sp1pointer used contains the domain for DNA binding as well as Sp1-Sp1interaction. As a result, these experiments did not demonstrateprecisely which type of Sp1 pointer is actually cleaving the DNA. Inorder to determine whether the LCR enhances the recruitment of thetethered Sp1, we repeated the experiment with Sp1 pointers that lackedthe DNA binding domain but contained domains A, B, C or A, B, C and D.Since these pointers cannot bind to the DNA directly, its recruitmentdepended on protein-protein interaction with Sp1 already bound to DNA.

[0244] As with the Sp1 pointer containing the DNA binding domain, thetethered Sp1 pointer cleaved within the β-globin promoter much moreefficiently when the promoter was joined to the LCR than to S phage DNA.This finding suggests that the LCR enhances the recruitment of tetheredSp1, and as a result, the formation of a multimeric Sp1 complexes on thepromoter. Such formation should amplify the activation signal from oneSp1 binding site by a factor of four, or possibly multiples of four, ifmultiple tetramers are recruited. The cleavage site of the Sp1 pointerswithout the DNA binding domain is near the TATA box, whereas that of theintact Sp1 pointer is near the CACCC box. Therefore, it is likely thatthe cleavage pattern of the intact Sp1 pointer reflects that of theDNA-bound Sp1. Further deletions of the glutamine rich domains A and Bor domain C resulted in no detectable cleavage, suggesting that thesedomains are important for the LCR enhanced recruitment.

[0245] 2. The CACCC and TATA Boxes are Important for Sp1 Recruitment.

[0246] The results discussed above demonstrate that the LCR plays animportant role in the recruitment of Sp1 pointer. To further dissect thesequences required for Sp1 recruitment, the following experimentsexamined the roles of the CACCC and TATA boxes in Sp1 recruitment. Adeletion of either box resulted in the loss of the LCR-enhancedrecruitment. Thus, the LCR and TFIID domains worked together inrecruiting Sp1 to the CACCC site. Since Sp1 has been shown to interactwith hTAFII55 and dTAFII110 in vitro, these are the most likelycomponents of TFIID to participate in the recruitment. Since therecruitment of TFIID is thought to be downstream of the recruitment oftranscription activators such as Sp1, these results indicate that theTFIID complex formed on the TATA box stabilizes the Sp1 complex. Sinceother general transcription factors such TFIIA and B also bind near theTATA box, they might also participate in this interaction. Sp1 pointerslacking the DNA binding domain, however, are not recruited to thepromoter region without the additional sequence upstream of the CACCCsite (UCAC) which contains binding sites for CAAT, NF1 and GATA-1. Thisfinding suggests that tethered Sp1, unlike DNA bound Sp1, needsadditional interactions with factors binding upstream of the CACCC site.

[0247] Each of the core regions of 5′ HS2, 3 and 4 contain multiplebinding sites for NFE-2, GATA-1, E and CACCC box proteins. The resultsdiscussed herein support the hypothesis that, through Sp1-Sp1 andSp1-GATA-1 interactions which are known to occur, LCR recruits Sp1 orperhaps more importantly, multimeric Sp1 complex to the CACCC site inthe promoter. This complex is further stabilized by interaction with thegeneral transcription machinery (e.g. TFIID) as well as transcriptionfactors bound upstream of the CACCC site. One clear implication of theseexperiments is that the binding affinity of a protein for a DNArecognition site by itself is not a good indicator of whether it bindsthe DNA in the complex milieu that exists in a cell; otherprotein-protein interactions such as that with other transcriptionfactors, the general transcription machinery, the chromatin componentsand nuclear matrix, and spatial compartmentalization within the nucleusare likely to play important roles in determining whether a protein isultimately recruited to a particular DNA sequence. The PINPOINTtechnology provides a method of analyzing the effect of suchinteractions.

[0248] 3. Individual Hypersensitive Sites Cooperate to Recruit Sp1Pointer.

[0249] The individual hypersensitive sites of the LCR actsynergistically to confer high position independent expression. Todetermine whether recruitment of Sp1 pointer to the β-globin promoter isdependent on such synergy, target DNAs having one or more hypersensitivesites deleted were tested for ability to recruit Sp1 pointer. Deletionof a single hypersensitivity site resulted in a significant reduction ofSp1 pointer recruitment to the promoter. A combination ofhypersensitivity sites 2 and 4 resulted in better recruitment than 2 and3 or 3 and 4, but not as well as 2, 3 and 4. This finding is consistentwith the notion that synergistic recruitment of Sp1 to the promoterplays a key role in the LCR mediated expression.

[0250] 4. Enhanced Sp1 Recruitment not a General Property of Enhancers.

[0251] To examine whether recruitment of transcription activators suchas Sp1 is a general property of enhancers, the LCR (p269) was comparedto three copies of SV40 enhancer (p399) in their abilities to activateβ-globin expression and to recruit Sp1. In transient transfection, theLCR and SV40 enhancers activate β-globin expression to similar levels.However, the SV40 enhancers do not appear to recruit Sp1 in the PINPOINTassay. Therefore, the strength of an enhancer does not always correlatewith its ability to recruit transcription activators such as Sp1. Thisobservation supports the hypothesis that one of the ways the LCR confersposition independence is by recruiting transcription activators such asSp1 to the promoter, which then participates in recruiting andstabilizing the general transcription factors near the TATA box.Consistent with this finding, the SV40 enhancer cannot confer positionindependence in transgenic mice. The results discussed herein, takentogether with recent evidence that certain transcription activatorspromote RNA elongation (Blair et al. (1996) EMBO J. 15:1658-65),cis-acting elements such as enhancers, LCRs or silencers are likely tofunction at various levels from recruitment of transcription activatorssuch as Sp1 to stabilization of general transcription machinery toprocessivity of RNA polymerase. These elements may also recruitchromatin organizing complexes such as SWI/SNF (Peterson and Tamkun(1995) Trends Biochem Sci 20: 143-6) and NURF (Tsukiyama et al. (1995)Cell 83: 1021-6) which then affect transcription factor recruitment andRNA elongation. Recent observations suggest that splicing factors arerecruited to the site of transcription. The PINPOINT technology makes itpossible to test whether the LCR plays a role in post-transcriptionalmodification such as splicing and polyadenylation by recruitingcomplexes involved in these processes.

[0252] This example demonstrates that the PINPOINT technology describedherein is useful for studying the interaction between protein andnucleic acids. The technology was used to analyze the role of theβ-globin LCR in the recruitment of transcription factors such as Sp1 tothe β-globin promoter in living MEL cells. The results of these studiesdemonstrate that, on a transfected plasmid, the LCR enhances Sp1recruitment and multimer formation on the β-globin promoter. Therecruitment requires the cooperation of other transcription factorsbound at the promoter region including TFIID. Based on these findings, amodel is proposed in which LCR-bound Sp1 and GATA-1, and perhaps otherfactors, recruit Sp1 to the CACCC site in the promoter. Since the TATAbox is also required for the LCR enhanced recruitment, TFIID and orTFIID associated factors are likely to stabilize recruited Sp1 ormultimeric complexes of Sp1. Such Sp1 recruitment appears to be mediatedthrough protein-protein interaction involving the glutamine-rich A and Bdomains and the C domain.

[0253] It is possible that the LCR also recruits chromatin remodelingcomplexes such as SWI/SNF or NURF to facilitate Sp1 recruitment if thepromoter region is in a nucleosome. Indeed, preliminary results indicatethat a mammalian SWI complex, BRG1, is recruited to the β-globinpromoter by the LCR. β-globin gene expression is very dependent on thesite of integration in transgenic mice, but is position independent whenthe gene is linked to the LCR. Such position dependence might be aresult of different chromatin structure and local concentration oftranscription factors throughout the genome. Thus, the LCR, byrecruiting chromatin remodeling complexes such as that of BRG1, appearsto open chromatin and recruit transcription activators, which in turnhelp recruit the general transcription machinery. Such LCR-mediatedrecruitment should help in keeping the promoter region nucleosome-freeregardless of the local chromatin structure.

[0254] The PINPOINT technology described herein can also be used toaddress additional questions regarding the role of the LCR in β-globingene expression. For example, one can use the technology to determinewhether the LCR enhances recruitment of transcription factors to theendogenous β-globin promoter. Also, the technology can be used todetermine whether additional CACCC box binding factors, such as EKLF andBKLF, also are recruited by the LCR and if so, which one is most likelyto bind the β-globin CACCC box. One can also use the PINPOINT technologyto determine whether the globin LCR recruits chromatin remodelingcomplexes such as BRG1 (or NURF) or histone modifying enzymes such ashistone acetyl transferases (Roth and Allis (1996) Cell 87: 5-8), bothof which should enhance recruitment of transcription factors.

[0255] In one embodiment, an in situ thermal cycler is used to amplifycleaved target nucleic acids. Where multiple sites are cleaved, thepositions on the nucleic acids are optionally assessed by microscopy, orby any of the PCR detection, cloning or sequencing protocols described.

Example 9

[0256] Preparation of FLASHPOINT Reagents

[0257] This Example describes the preparation of a reagents that areuseful in the FLASHPOINT methods of the invention. These reagentsinclude an IMMUNOPOINTER, which consists of a micrococcal nucleasetethered to an immunoglobulin, as well as a molecular beacon conjugatedto an antibody.

[0258] Preparation of an IMMUNOPOINTER

[0259] An expression vector for a fusion protein containing the flexiblepolypeptide linked to the micrococcal nuclease was constructed asfollows. The coding region of the micrococcal nuclease was amplified bypolymerase chain reaction using primers JC373(5′-TGAAGACGAATTCACCGGTGCAACTTCAACTAAAAAATTACATAAAGAACCTGCGACTTTAATTAAAGCGATTGATGGTGAGACGGT-3′;(SEQ ID NO:14)) and JC374(5′-GACGACGGATCCGGAAGCGGCCGCTTGACCTGAATCAGCGTTGTC-3′; (SEQ ID NO:15)),which introduced SgrA1 and BspE1 cleavage sites in the 5′ and 3′ ends,respectively. Because JC373 included an ASP21 to GLU21 mutation, theamplified micrococcal nuclease also contained this mutation. Theamplified fragment was cleaved with SgrA1 and BspE1 and cloned intoSgrA1 and BspE1 sites of p461, which is a pBluescript2-based vectorcontaining a humanized micrococcal nuclease (MNase) structural gene intowhich one can clone a guide domain. To construct a chimericendonuclease, a guide domain coding region is placed upstream of theMNase structural gene and downstream of a CMV promoter. A polylinker ispresent at position 1245 for insertion of the guide domain fragment. Thenucleotide sequence of p461 is provided as SEQ ID NO:19 in commonlyassigned U.S. patent application Ser. No. 08/825,664, which was filed onApr. 3, 1997.

[0260] The resulting plasmid (p566) was cleaved with SmaI and a linker(JC375; 5′-GCAACCCATGGGTTGC-3′; (SEQ ID NO:16)) was ligated to the bluntends. Ligation of the linker introduced an NcoI site and a cysteineresidue in frame immediately 5′ to the flexible polypeptide. A DNAfragment encoding the flexible polypeptide-micrococcal nuclease fusionprotein (hence forth called the nuclease tail) was isolated by cleavingthe linker-ligated fragment with NcoI and NotI and cloned into abacterial expression vector pET22b(+) (Novagen). The resulting plasmid(p616) was introduced into BL21(DE3) competent E. coli cells (Novagen)for the expression of the micrococcal nuclease tail containing the GLU21mutation (Serpersu et al. (1987) Biochemistry 26: 1289-300).

[0261] In order to express micrococcal nuclease tail with ASP21 (wildtype), GLU21 in p616 was converted to ASP21 by PCR amplifying the regioncontaining the codon for amino acid 21 in p616 with primers JC396(5′-TGCGGGGACTCGAGTCTGCA-3′; (SEQ ID NO:17)) and JC397(5′-GACCTTTGTACATTAATTTAACCGTATCACCATCAATCGCT-3′; (SEQ ID NO:18)),cleaving the amplified fragment with SgrA 1 and BsrG 1 and cloning itinto p616. The resulting plasmid, p684, was introduced into BL21(DE3)competent E. coli cells for the expression of the wild type micrococcalnuclease tail. The amino acid numbering scheme is based on that ofmicrococcal nuclease secreted from Staph. aureus (Shortle (1983) Gene22: 181-9).

[0262] BL21(DE3) E. coli cells transformed with the expression vectorsdescribed above were grown in LB and induced with IPTG. After induction,cells were collected by centrifugation at 5000×g for 5 min. The cellpellet was resuspended in 1×binding buffer (5 mM imidazole, 800 mM NaCland 20 mM Tris-HCl pH 7.9) and sonicated. The lysate was centrifuged at39,000×g for 20 minutes to remove debris. The nuclease tail, whichcontains 6 histidine residues at the carboxy terminus was then purifiedby Ni²⁺ column chromatography.

[0263] Crosslinking the Nuclease Tail to Antibody (IMMUNOPOINT).

[0264] The nuclease tail, purified as described above, was reduced byincubating it with 100 mM DTT in PBS (phosphate-buffered saline) at 37°C. for 30 minutes. Reduced nuclease tail was desalted into PBScontaining 5 mM EDTA. The antibody (0.5-1.0 mg) to be crosslinked to thenuclease tail was treated with 50-500 mg SMCC (succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, Pierce ChemicalCompany, Rockford, Ill.) in 500 ml of PBS for 30 minutes at 37° C. TheSMCC treated antibody was desalted into PBS. Desalted nuclease tail (atgreater than four fold excess) and SMCC crosslinked antibody wereincubated together at 4° C. overnight. The antibody crosslinked to thenuclease tail (IMMUNOPOINTER) was further purified with Ni²⁺ columnchromatography and or protein A/G affinity matrix (Pierce ChemicalCompany). Other proteins may be crosslinked to the nuclease tail in asimilar manner. The crosslinked nuclease is activated by adding 1 mMCaCl₂ for 1 to 10 minutes.

[0265] Attachment of Molecular Beacon to Antibody

[0266] Equal concentrations of an antibody and a molecular beaconmolecule (synthesized by CyberSyn, Lenni, Pa.) were mixed with BS3(Bis(sulfosuccinimidyl) suberate, Pierce Chemical Company) in pH 7.5PBS. When the protein concentration is above 5 mg/ml, 10-fold molarexcess of the crosslinker (BS3) over the protein was used; when theprotein concentration was below 5 mg/ml, 20- to 50-fold molar excess ofthe crosslinker was used. The reaction mixture was incubated at roomtemperature for 30 minutes or on ice for 2 hours. The reaction isquenched for 30 minutes with a Tris or glycine buffer, or with a buffercontaining lysine (20-50 mM). Other proteins may be crosslinked to abeacon molecule in a similar manner.

[0267] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference in its entirety for allpurposes. Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the following claims.

What is claimed is:
 1. A method of cleaving a target nucleic acid in acell with a chimeric guide-endonuclease fusion molecule, the methodcomprising: (i) providing a cell comprising the target nucleic acid anda chimeric guide molecule-endonuclease fusion molecule; and, (ii)permitting the fusion molecule to cleave the target nucleic acid,thereby producing a cleaved target nucleic acid with a site of cleavage.2. The cell of claim 1, wherein the cell comprises a nucleic acid whichencodes the chimeric guide molecule-endonuclease fusion molecule, whichfusion molecule is a protein, and the method further comprises the stepof expressing the chimeric guide molecule-endonuclease fusion moleculein the cell, thereby providing the cell comprising the target nucleicacid and a chimeric guide molecule-endonuclease fusion molecule.
 3. Themethod of claim 2, further comprising transducing the cell with thechimeric nucleic acid encoding the chimeric guide molecule-endonucleasefusion molecule.
 4. The method of claim 2, further comprisingco-transducing the cell with the chimeric nucleic acid encoding thechimeric guide molecule-endonuclease fusion molecule and the targetnucleic acid, and optionally comprising the step of transducing the cellwith a marker nucleic acid.
 5. The method of claim 2, further comprisingthe steps of transducing the cell with a nucleic acid encoding aselectable component, and selecting transduced cells using theselectable component, wherein expression of the selectable componentcorresponds to transduction of the cell by the chimeric nucleic acidencoding the guide molecule-endonuclease chimeric molecule.
 6. Themethod of claim 1, further comprising detecting the cleaved nucleicacid.
 7. The method of claim 1, further comprising amplifying thecleaved target nucleic acid.
 8. The method of claim 1, furthercomprising the steps of amplifying the cleaved target nucleic acid anddetecting the amplified target nucleic acid.
 9. The method of claim 1,further comprising transducing the cell with the target nucleic acid.10. The method of claim 1, further comprising the step of detecting thecleaved nucleic acid, wherein the target nucleic acid is detected usinga technique selected from the group consisting of nested PCR, Southernblotting, northern blotting, and, cloning and sequencing the targetnucleic acid.
 11. The method of claim 1, wherein the cell is embedded inagarose to reduce shearing of the target nucleic acid.
 12. The method ofclaim 1, wherein the target nucleic acid is selected from the groupconsisting of a genomic nucleic acid with a known sequence ofnucleotides, a genomic nucleic acid with an unknown sequence ofnucleotides, a plasmid with a sequence of known nucleotides, a plasmidwith a sequence of unknown nucleotides, an RNA with a sequence of knownnucleotides, and an RNA with an unknown sequence of nucleotides.
 13. Themethod of claim 1, wherein the chimeric guide-molecule endonucleasefusion molecule is a calcium inducible protein, and the method furthercomprises the step of adding exogenous calcium to the cell, therebyinducing the protein to cleave the target nucleic acid in step (ii). 14.The method of claim 1, wherein the endonuclease is derived frommicrococcal nuclease.
 15. The method of claim 1, wherein the guidedomain is derived from the group of proteins consisting of a DNA bindingprotein, an RNA binding protein, a protein which binds to a DNA bindingprotein, a protein which binds to an RNA binding protein, an antibodyprotein which binds to a DNA binding protein, a first antibody proteinwhich binds to a second antibody protein, an antibody protein whichbinds to an RNA binding protein, and, a guide nucleic acid whichhybridizes to the target nucleic acid.
 16. The method of claim 1,wherein cleavage of the target nucleic acid by the guide endonucleasechimera produces a 3′ phosphate and a 5′ hydroxyl at the site ofcleavage.
 17. The method of claim 1, further comprising the step ofligating an oligonucleotide to the cleaved target nucleic acid.
 18. Themethod of claim 1, further comprising the steps of: (a) making a firstdouble-stranded target DNA and, optionally, a second double-strandedtarget DNA which correspond to each side of the cleavage site; (b)ligating a first oligonucleotide comprising a first class IISrestriction site to the first double-stranded target DNA, therebyproducing a first ligated target DNA, the first oligonucleotideoptionally further comprising a first class I restriction site and afirst detectable label; (c) optionally, ligating a secondoligonucleotide comprising a second class IIS restriction site to thesecond double-stranded target DNA, thereby producing a second ligatedtarget DNA, the second oligonucleotide optionally further comprising afirst class I restriction site and a second detectable label; (d)cleaving the first ligated target DNA and, optionally, cleaving thesecond ligated target DNA with the first and, optionally, the secondclass IIS restriction enzymes; and (e) isolating the first and,optionally, the second ligated target DNAs, optionally by capture of thefirst and second detectable label and cleavage of the captured labelwith the first and second class I restriction enzyme.
 19. The method ofclaim 18, further comprising the steps of: (f) ligating the first andsecond ligated target DNAs to form a ligated target site; and optionallycomprising the steps of: (g) concatemerizing the ligated target site toproduce a concatemerized target site; (h) cloning the concatemerized thetarget site into a nucleic acid vector to produce a cloned target site;and, (i) sequencing the cloned target site.
 20. The method of claim 19,further comprising the steps of: (j) 3′ dephosphorylation of the 3′phosphate at the site of cleavage, thereby producing a 3′dephosphorylated cleavage end; (k) extending the dephosphorylatedcleavage end, thereby producing an extended 3′ end; and, (l) PCRamplifying the extended 3′ end.
 21. The method of claim 1, wherein thetarget nucleic acid is a plasmid nucleic acid and the chimeric guidedomain is a calcium inducible polypeptide domain, the method furthercomprising the steps of: (a) permeabilizing the cell and treating thecell with calcium, thereby inducing the calcium inducible chimericmolecule, which molecule cleaves at least one strand of the plasmid,leaving a 3′ phosphate and a 5′ hydroxyl the site of cleavage; (b)isolating plasmid nucleic acids from the cell, including the cleavedplasmid, thereby producing isolated plasmid nucleic acids, (c) primerextending the cleaved plasmid using a primer extension primer which iscomplementary to a strand of the plasmid comprising the cleavage site,thereby producing a double-stranded blunt end at the site of cleavage;(d) ligating a trapping oligonucleotide to the double-stranded bluntend; (e) PCR amplifying the cleaved plasmid using a PCR reaction mixturecomprising an oligonucleotide which hybridizes to the trappingoligonucleotide, and, optionally, the primer extension primer, therebyamplifying the nucleic acid; and, optionally, further comprising thestep of: (f) cloning the guide molecule-chimera, thereby producing achimeric nucleic acid, wherein an endonuclease cleavage domain of thechimeric molecule is calcium inducible, cleaves a single strand of theplasmid, and produces a 3′ phosphate and a 5′ hydroxyl at the site ofcleavage.
 22. The method of claim 21, further comprising the steps of:(g) 3′ dephosphorylating a 3′ phosphate at the site of cleavage, therebyproducing a 3′ dephosphorylated cleavage end; (j) extending thedephosphorylated cleavage end, thereby producing an extended 3′ end;and, (k) PCR amplifying the extended 3′ end.
 23. The method of claim 21,wherein an internal PCR primer is used in step (e.) to prime the PCR.24. The method of claim 21, further comprising detecting the PCRamplified nucleic acid.
 25. The method of claim 1, wherein cleavage ofthe target nucleic acid by the guide molecule-endonuclease chimeraproduces a 3′ phosphate and a 5′ hydroxyl at the site of cleavage,further comprising one or more step selected from the steps of: (a) 5′phosphorylating the cleaved nucleic acid at the site of cleavage toproduce a 5′ phosphorylated site; (b) 3′ dephosphorylating the cleavednucleic acid at the site of cleavage; (c) extending the 3′ end of thecleavage site with a terminal transferase enzyme; (d) extending the 3′end of the cleavage site by ligating a 3′ extension oligonucleotide tothe cleaved nucleic acid; (e) primer extending the cleaved targetnucleic acid using a primer extension primer which is complementary to astrand of the nucleic acid comprising the cleavage site, therebyproducing a double-stranded blunt end at the site of cleavage; (f)extending the 5′ end by ligating a 5′ extension oligonucleotide to thecleaved target DNA; (g) PCR amplifying target nucleic acid using aprimer complementary to the 5′ extension oligonucleotide; and, (h)performing nested PCR on amplified nucleic acids.
 26. The method ofclaim 1, wherein cleavage of the target nucleic acid by the guidemolecule-endonuclease chimera produces a 3′ phosphate and a 5′ hydroxylat the site of cleavage, further comprising one or more step selectedfrom the steps of: (a) 5′ phosphorylating the cleaved nucleic acid atthe site of cleavage to produce a 5′ phosphorylated site; (b) 3′dephosphorylating the cleaved nucleic acid at the site of cleavage; (c)extending the 3′ end of the cleavage site with a terminal transferaseenzyme; (d) extending the 3′ end of the cleavage site by ligating a 3′extension oligonucleotide to the cleaved nucleic acid; (e) primerextending the cleaved target nucleic acid using a primer extensionprimer which is complementary to a strand of the nucleic acid comprisingthe cleavage site, thereby producing a double-stranded blunt end at thesite of cleavage; (f) extending the 5′ end by ligating a 5′ extensionoligonucleotide to the cleaved target DNA; (g) PCR amplifying targetnucleic acid using a primer complementary to the 5′ extensionoligonucleotide; and, (h) performing nested PCR on amplified nucleicacids.
 27. The method of claim 1, wherein the target nucleic acid is agenomic DNA, the chimeric molecule is calcium inducible, and the methodfurther comprises the steps of: (a) co-transducing the cell with amarker vector and a chimeric nucleic acid vector encoding the chimericguide molecule, and culturing the cell under conditions which permitexpression of a marker encoded by the marker vector and the chimericguide molecule, thereby producing a marked cell which expresses themarker and the chimeric guide molecule; (b) isolating the marked cell,thereby providing an isolated cell; (c) permeabilizing the isolated cellwith a mild detergent and treating the isolated cell with calcium,thereby inducing the calcium inducible chimeric molecule, which moleculecleaves at least one strand of the target nucleic acid, leaving a 3′phosphate and a 5′ hydroxyl the site of cleavage; and, (d) furtherperforming one or more step selected from the steps of: (e) 5′phosphorylating the 5′ hydroxyl of step (c), thereby providing a 5′phosphate site on the cleaved nucleic acid for primer extension; (f)ligating a trapping oligonucleotide to the 5′ phosphate site to producea target-linker nucleic acid; (g) 3′ dephosphorylating the 3′ phosphateat the site of cleavage; (h) 3′ end extending the 3′ cleavage site witha terminal transferase enzyme to produce a first extended-target nucleicacid; (i) 3′ end extending the 3′ cleavage site with by ligation of anoligonucleotide to produce a second extended-target nucleic acid; (j)PCR amplifying the target-linker nucleic acid of step (f), to produce anamplified target-linker nucleic acid; (k) 3′ end extension of theamplified target-linker nucleic acid; (l) PCR amplification of the firstextended-target nucleic acid of step (h) to produce a first amplifiedextended-target nucleic acid; (m) PCR amplification of the secondextended target nucleic acid of step (i) to produce a second amplifiedextended-target nucleic acid; and, (n) detection of a nucleic acidselected from the group consisting of the first amplifiedextended-target nucleic acid of step (l), the second amplifiedextended-target nucleic acid of step (m) and the amplified target-linkernucleic acid of step (j).
 28. The method of claim 1, wherein the targetnucleic acid is an RNA, wherein cleavage of the RNA by the guidemolecule-endonuclease chimera produces a 3′ phosphate and a 5′ hydroxylat the site of cleavage and the method further comprises the steps of:(a) isolating RNA from the cell, thereby producing isolated RNA; (b)coupling an RNA terminator to 3′ hydroxyls present in the isolated RNA;and, (c) dephosphorylating the 3′ phosphate to produce adephosphorylated target RNA, using an enzyme selected from the groupconsisting of a kinase enzyme, and a phosphatase enzyme.
 29. The methodof claim 28, further comprising one or more steps selected from thesteps consisting of: (d) ligating a first oligonucleotide to thedephosphorylated target RNA, which first oligonucleotide optionallycomprises a binding site for a class IIS restriction enzyme; (e)ligating a second oligonucleotide to the dephosphorylated target RNAwhich second oligonucleotide optionally comprises a binding site for aclass IIS restriction enzyme; (f) reverse transcribing the cleavedtarget nucleic acid, optionally using a reverse transcription primerwhich hybridizes to the first oligonucleotide of step (d) to prime thereverse transcription reaction, thereby producing a reverse transcribednucleic acid; (g) treating the reverse transcribed nucleic acid of step(e) with a first enzyme with RNAse H activity, and a second enzyme withDNA polymerase activity to make a double-stranded target DNA; (h)cleaving the double-stranded DNA with a restriction enzyme, therebyproducing a restricted target DNA; (i) ligating a third oligonucleotideto the restricted target DNA; (j) isolating the restricted target DNA,thereby producing an isolated restricted target DNA; (k) PCR amplifyingthe isolated restricted target DNA, thereby producing amplifiedrestricted target DNA; and, (l) cloning the restricted amplified targetDNA.
 30. The method of claim 28, further comprising one or more stepsselected from the steps consisting of: (d) ligating a firstoligonucleotide to the dephosphorylated target RNA, which firstoligonucleotide optionally comprises a binding site for a class IISrestriction enzyme; (e) ligating a second oligonucleotide to thedephosphorylated target RNA which second oligonucleotide optionallycomprises a binding site for a class IIS restriction enzyme; (f) reversetranscribing the cleaved target nucleic acid, optionally using a reversetranscription primer which hybridizes to the first oligonucleotide ofstep (d) to prime the reverse transcription reaction, thereby producinga reverse transcribed nucleic acid; (g) treating the reverse transcribednucleic acid of step (e) with a first enzyme with RNAse H activity, anda second enzyme with DNA polymerase activity to make a double-strandedtarget DNA; (h) cleaving the double-stranded DNA with a restrictionenzyme, thereby producing a restricted target DNA; (i) ligating a thirdoligonucleotide to the restricted target DNA; (j) isolating therestricted target DNA, thereby producing an isolated restricted targetDNA; (k) PCR amplifying the isolated restricted target DNA, therebyproducing amplified restricted target DNA; and, (l) cloning therestricted amplified target DNA.
 31. The method of claim 1, wherein themethod further comprises one or more step selected from the steps of:(a) random primer extending the cleaved target nucleic acid; (b)ligating a blocking oligonucleotide to the cleaved target nucleic acid;(c) 5′ phosphorylating the cleaved target nucleic acid; (d) cleaving thetarget nucleic acid with a restriction enzyme; (e) cleaving an amplifiedtarget nucleic acid with a restriction enzyme; (f) trapping cleavedtarget DNA to produce trapped target DNA; (g) ligating a reachingoligonucleotide to the trapped target DNA; (h) amplifying the target DNAusing PCR; (i) blocking a 3′OH terminal of the cleaved target nucleicacid with a terminal transferase; (j) blocking a 3′OH terminal of thecleaved target nucleic acid with a blocking oligonucleotide; (k) 3′dephosphorylating the cleaved target nucleic acid; (l) 3′ end extendingthe cleaved target nucleic acid with a terminal transferase enzyme; (m)3′ end extending the cleaved target nucleic acid by ligating anoligonucleotide onto the 3′ end of a cleaved target nucleic acid; (n)primer extending the cleaved target nucleic acid; (o) cloning the guidemolecule-chimera; and, (p) combining one or more of the preceding steps(a)-(o).
 32. The method of claim 1, wherein the method further comprisesone or more step selected from the steps of: (a) random primer extendingthe cleaved target nucleic acid; (b) ligating a blocking oligonucleotideto the cleaved target nucleic acid; (c) 5′ phosphorylating the cleavedtarget nucleic acid; (d) cleaving the target nucleic acid with arestriction enzyme; (e) cleaving an amplified target nucleic acid with arestriction enzyme; (f) trapping cleaved target DNA to produce trappedtarget DNA; (g) ligating a reaching oligonucleotide to the trappedtarget DNA; (h) amplifying the target DNA using PCR; (i) blocking a 3′OHterminal of the cleaved target nucleic acid with a terminal transferase;(j) blocking a 3′OH terminal of the cleaved target nucleic acid with ablocking oligonucleotide; (k) 3′ dephosphorylating the cleaved targetnucleic acid; (l) 3′ end extending the cleaved target nucleic acid witha terminal transferase enzyme; (m) 3′ end extending the cleaved targetnucleic acid by ligating an oligonucleotide onto the 3′ end of a cleavedtarget nucleic acid; (n) primer extending the cleaved target nucleicacid; (o) cloning the guide molecule-chimera; and, (p) combining one ormore of the preceding steps (a)-(o).
 33. The method of claim 1, whereinthe method further comprises fixing and permeabilizing the cell.
 34. Amethod of screening test nucleic acids for in vivo cleavage sites whichare cleaved by a chimeric guide molecule, comprising: (A) providing acell comprising a chimeric nucleic acid encoding the chimeric guidemolecule and a test nucleic acid; (B) permitting the chimeric nucleicacid to be expressed in the cell, thereby producing chimeric guidemolecule in the cell; (C) incubating the cell under conditions in whichthe guide molecule is active, and, (D) determining whether the chimericguide molecule cleaves the test nucleic acid, thereby determiningwhether the test nucleic acid comprises an in vivo cleavage site for thechimeric guide molecule.
 35. The method of claim 34, wherein the testnucleic acid encodes a promoter sequence operably linked to a reportergene, and the method further comprises detection of the presence orabsence of reporter gene expression, which expression is an indicatorfor whether the test nucleic acid comprises an in vivo cleavage site forthe chimeric guide molecule.
 36. The method of claim 34, furthercomprising the step of co-transducing a cell with a first plasmidencoding the target nucleic acid and a second plasmid encoding thechimeric guide molecule.
 37. The method of claim 34, further comprisingtransducing the cell with a second test nucleic acid and determiningwhether the chimeric guide molecule cleaves the second test nucleicacid, thereby determining whether the second test nucleic acid comprisesan in vivo cleavage site for the chimeric guide molecule.
 38. The methodof claim 34, further comprising, in parallel with steps (A)-(D),performing the steps of: (E) providing a second cell comprising a secondchimeric nucleic acid encoding a second chimeric guide molecule and asecond test nucleic acid; (F) permitting the second chimeric nucleicacid to be expressed in the second cell, thereby producing the secondchimeric guide molecule in the second cell; (G) incubating the secondcell under conditions in which the second guide molecule is active, and,(H) determining whether the second chimeric guide molecule cleaves thesecond test nucleic acid, thereby determining whether the second testnucleic acid comprises an in vivo cleavage site for the chimeric guidemolecule.
 39. The method of claim 31, wherein the cell further comprisesa control nucleic acid which is cleaved by the chimeric guide molecule,and the method further comprises the steps of: (E) permitting thecontrol nucleic acid to be cleaved in the cell during step (C); and, (F)comparing the rate of cleavage of the test nucleic acid and the controlnucleic acid, thereby determining the affinity of the guide molecule forthe test nucleic acid, as compared to the control nucleic acid.
 40. Amethod of detecting a chimeric guide endonuclease molecule modulatingagent, comprising the steps of: providing a cell comprising a testnucleic acid, which cell expresses a chimeric guide-endonucleasemolecule; contacting the cell with the modulating agent; and, measuringthe rate of cleavage of the test nucleic acid by the chimeric guideendonuclease molecule in the presence of the agent.
 41. The method ofclaim 40, wherein the target nucleic acid is a promoter sequence whichis bound by a transcription factor.
 42. The method of claim 40, furthercomprising the step of comparing the rate of cleavage of the testnucleic acid in the presence of the agent to the rate of cleavage of thetest nucleic in an absence of the agent.
 43. The method of claim 40,wherein the cell further comprises a second test nucleic acid bindingsite and the method further comprises the steps of measuring the rate ofcleavage of the second test nucleic acid in the presence of the agent.44. The method of claim 40, further comprising the steps of: providing asecond cell comprising a second test nucleic acid; contacting the secondcell with the modulating agent; and, measuring the rate of cleavage ofthe second test nucleic acid by the chimeric molecule in the presence ofthe agent.
 45. The method of claim 44, wherein the second cell iscontacted with the modulating agent in parallel with contacting thefirst cell with the modulating agent.
 46. A method of detecting whethera first molecule is in close proximity to a second molecule, the methodcomprising the steps of: attaching a molecular beacon to the firstmolecule, wherein the molecular beacon comprises an oligonucleotide towhich is attached a fluorophore and a quencher; attaching anendonuclease moiety to the second molecule; and determining whether thefirst molecule is in close proximity to the second molecule by detectingwhether fluorescence is emitted by the fluorophore, wherein fluorescenceemission is indicative of cleavage of the oligonucleotide by theendonuclease moiety, thereby causing separation of the fluorophore andthe quencher.
 47. The method of claim 46, wherein either or both of thefirst molecule and the second molecule are located in a cell.
 48. Themethod of claim 46, wherein either or both of the first molecule and thesecond molecule are located in an organism.
 49. The method of claim 46,wherein the target molecule is present in a tissue sample.
 50. Themethod of claim 46, wherein the molecular beacon is covalently attachedto the first molecule.
 51. The method of claim 46, wherein the molecularbeacon is noncovalently attached to the first molecule.
 52. The methodof claim 51, wherein the molecular beacon is attached to a bindingmoiety which binds to the first molecule.
 53. The method of claim 52,wherein the binding moiety is an antibody.
 54. The method of claim 46,wherein the endonuclease moiety is covalently attached to the secondmolecule.
 55. The method of claim 46, wherein the endonuclease moiety isnoncovalently attached to the second molecule.
 56. The method of claim55, wherein the endonuclease moiety comprises a binding moiety whichbinds to the second molecule.
 57. The method of claim 56, wherein thebinding moiety is an antibody.
 58. The method of claim 46, wherein thefirst molecule and the second molecule are separated by about tennanometers or less.
 59. The method of claim 58, wherein the firstmolecule is in contact with the second molecule.
 60. The method of claim46, wherein the endonuclease moiety comprises a calcium-inducibleendonuclease, and the method further comprises the step of contactingthe endonuclease moiety with calcium.
 61. The method of claim 46,wherein the first molecule comprises a first member of a binding pairand the second molecule comprises a second member of the binding pair,and wherein the binding pair is selected from the group consisting of:enzyme:substrate, hormone:ligand, drug:receptor, protein:protein,protein/modifier, protein:nucleic acid; and nucleic acid:nucleic acid.62. The method of claim 46, wherein the emission of fluorescence isdetected by fluorescent microscopy or fluorometry.
 63. A method ofdetecting a target molecule, the method comprising the steps of:contacting the target molecule with a chimeric endonuclease which bindsto the target molecule; contacting the chimeric endonuclease with amolecular beacon comprising an oligonucleotide to which is attached afluorophore and a quencher; and detecting the presence of a fluorescentsignal which results from cleavage of the oligonucleotide by theendonuclease, thereby allowing separation of the quencher from thefluorophore.
 64. The method of claim 63, wherein the target molecule ispresent in a cell.
 65. The method of claim 63, wherein the targetmolecule is present in a tissue sample.
 66. The method of claim 63,wherein the fluorescent signal is integrated over time.
 67. The methodof claim 63, wherein the chimeric endonuclease comprises acalcium-inducible endonuclease moiety, and the method further comprisesthe step of contacting the endonuclease moiety with calcium.
 68. Themethod of claim 63, wherein the chimeric endonuclease binds directly tothe target molecule.
 69. The method of claim 68, wherein the targetmolecule is a nucleic acid and the chimeric endonuclease comprises anucleic acid binding domain.
 70. The method of claim 63, wherein thechimeric endonuclease binds indirectly to the target molecule.
 71. Themethod of claim 70, wherein a primary binding moiety binds to the targetmolecule and the chimeric endonuclease binds to the primary bindingmoiety.
 72. The method of claim 71, wherein the primary binding moietyis an antibody that binds to the target molecule and the chimericendonuclease comprises a moiety that binds to the antibody.